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DNA REPLICATION-in all cells, DNA sequences should be maintained and replicatedwith high fidelity (mutation rate, approximately 1 nucleotidechange per 109 nucleotides each time the DNA is replicated, isroughly the same for organisms as different as bacteria andhumans).-the sequence of the human genome (approximately 3 109

nucleotide pairs) is changed by only about 3 nucleotides each time acell divides.-this allows most humans to pass accurate genetic instructions fromone generation to the next, and also to avoid the changes in somaticcells that lead to cancer

Why Study DNA Replication?

1) Understanding Cancer-- the uncontrolled cell division (DNA replication).2) Understanding Aging--cells are capable of a finite number of doublings.

3) Understanding Diseases associated with defects in DNA repair.

DNA REPLICATION, REPAIR and RECOMBINATION

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1) CancerCells are carefully controlled in the number of cell doublings that they arecapable of as well as when cell division will occur. In cancer the control ofinitiation of replication is lost

2) AgingFor example, fibroblast cells (connective tissue) in culture will double forabout 50 generations. Then they enter senescence. Senescent cells are nolonger capable of dividing yet remain metabolically active. In addition,they exhibit changes in form and function, which may lead to age-related

changes such as the difference between the supple skin of a child and thewrinkled skin of the elderly.3) DNA repair diseases

There are several diseases that cause premature aging or sensitivity to UVlight.

Examples include:

a) Bloom Syndrome, a cancer-prone genetic disorder due to genetic instability inthe form of increased frequencies of breaks of the chromosomes.

b) Xeroderma Pigmentosum, a human DNA repair deficiency syndrome leadingto predisposition to sun-light-induced skin cancer.

c)Werner Syndrome, a premature aging disease that begins in adolescence orearly adulthood and results in the appearance of old age by 30-40 years of

age.

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Xeroderma patient

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KEY CONCEPTS:

Proteins interact with DNA in all biological activities involvingDNA.

DNA must be unwound to replicate. Topoisomerases catalyze changes in supercoiled state of DNA. DNA replication has three distinct phases (initiation, elongation,

and termination). Termination is different at telomeres ofeucaryotic chromosomes

DNA replication is very accurate (1x10-8 mistakes/base).DNA molecules can recombine if they have similar sequences. Mutations have several causes and involve base sequence changes.

DNA repair corrects errors using highly evolved correctionsystems.

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Three general features of Chromosomal replication:1. DNA Replication Is Semiconservative

1958: Meselson and Stahl: DNA

Replication is Semiconservative

*(in both prokaryotes & eukaryotes)

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2. Most DNA Replication Is Bidirectional

Figure 12-2. Three mechanisms of DNA strand growth that are consistent with

semiconservative replication.The third mechanismbidirectional growth of both strandfrom a single originappears to be the most common in both eukaryotes and prokaryotes.

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Because of the anti-parallel structure of the DNA duplex, new DNA must besynthesized in both the 5 to 3 and 3 to 5 directions overall.However all known DNA polymerases synthesize DNA in the 5 to 3 directiononly.

The solution is semidiscontinuous DNA replication.

Leading Strand-replicates continuously

Lagging Strand-replicates discontinuously-consists of OkasakiFragments (ss DNA chains1000-2000 nucleotides long,primed by very short RNA

primers) which need to bejoined by DNA ligase-the parental strand forms atrombone structure

RNA primers

The Leading and Lagging

Strands Are Synthesized

Concurrently

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3. DNA Replication Begins at Specific Chromosomal Sites-DNA synthesis is initiated at special regions called replication origins. Abacterial chromosome has one origin, whereas each eukaryotic

chromosome has many (hundreds or even thousands).Close-up of a replication forkorigin of replication

Bubble

Parental (template) strandDaughter (new) strand

Replication fork

Two daughter DNA molecules

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Figure 5.14. Origin of replication inE. coli Replication initiates

at a unique site on the E. coli chromosome, designated theorigin (ori)

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Figure 5.15. Replication origins in eukaryotic chromosomes Replicationinitiates at multiple origins (ori), each of which produces two replication

forks.

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Three Common Features of Replication Origins

1. replication origins are unique DNA segments that contain multiple

short repeated sequences

2. these short repeat units are recognized by multimeric origin-binding

proteins.

3. origin regions usually contain an AT-rich stretch

*Origin-binding proteins control the initiation of DNA replication bydirecting assembly of the replication machinery to specific sites on theDNA chromosome.

Replicon - region of DNA served by one replication origin.

Three types of replication origins:1. E. coli oriC2. yeast autonomously replicating sequences (ARS)3. simian virus 40 (SV40) origin.

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1. oriCis an 240-bp DNA segment present at the start site for replicationof E. coli chromosomal DNA-contain repetitive 9-bp and AT-rich 13-bp sequences, referred toas 9-mers (dnaA boxes) and 13-mers, respectively.

Figure 12-5. Consensus sequence of the minimalbacterial replication origin

*these are binding sites for the DnaA protein that initiatesreplication.

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2. Yeast Autonomously Replicating Sequences- has multiple origins of replication-confers on a plasmid the ability to replicate in yeast and is arequired element in yeast artificial chromosomes-a 15-bp segment, designated element A, stretching fromposition 114 through 128 which contains an 11-base-pair ARSconsensus sequence (ACS), which is the specific binding site ofthe origin replication complex (ORC).-three additional elements (B1, B2, and B3) are individually not

essential but together contribute to ARS function.

Figure 5.17. A yeastARS element

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3. SV40 Replication Origin-A 65-bp region in the SV40 chromosome is sufficient to promoteDNA replication both in animal cells and in vitro.

-three segments of the SV40 origins are required for activity-initiated by a virus-encoded protein (called T antigen) that bindsto the origin and also acts as a helicase.

The DNA Replication MachineryDNA Polymerases

DNA polymerases are unable to melt duplex DNA (i.e., break theinterchain hydrogen bonds) in order to separate the two strands that are tobe copied.

All DNA polymerases so far discovered can only elongate apreexisting DNA or RNA strand, the primer; they cannot initiate chains.

The two strands in the DNA duplex are opposite (53 and 35)in chemical polarity, but all DNA polymerases catalyze nucleotide additionat the 3-hydroxyl end of a growing chain, so strands can grow only in the53 direction.

*In this section, we describe the cell's solutions to the unwinding,priming, and directionality problems resulting from the structure of DNAand the properties of DNA polymerases

T bl 2 S f h P i R i d f R li i

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E. coliprotein Eukaryotic protein Function

DnaA ORC proteins Recognition of origin of replication

Gyrase Topoisomerase I/II Relieves positive supercoils ahead of

replication fork

DnaB Mcm DNA helicase that unwinds parentalduplex

DnaC ? Loads helicase onto DNA

SSB RFA Maintains DNA in single-stranded state

-complex RFC Subunits of the DNA polymerase

holoenzyme that load the clamp onto

the DNApol III core pol / Primary replicating enzyme; synthesizes

entire leading strand and Okazaki

fragments; has proofreading capability

subunit PCNA Ring-shaped subunit of DNA polymerase

holoenzzyme that clamps replicating

polymerase to DNA;works with pol III in

E. coli and pol or in eukaryotes

Primase Primase Synthesizes RNA primers

- pol Synthesizes short DNA oligonucleotides

as part of RNA-DNA primer

DNA ligase DNA ligase Seals Okazaki fragments into continuous

strandpol I FEN-1 Removes RNA primers; pol I ofE.coli

Table 2. Some of the Proteins Required for Replication

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Figure 12-7. Model of initiation ofreplication atE. coli oriC.

DnaA Protein Initiates

Replication in E. coli

DnaB Is an E. coliHelicase That

Melts Duplex DNA

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Replication overview

Must maintain integrity of the DNA sequencethrough successive rounds of replication

Need to:

unwind DNA, add an RNA primer, find anappropriate base, add it to the growing DNAfragment, proofread, remove the initial primer,fill in the gap with DNA, ligate fragmentstogether

All of this is fast, about 100 bp/second

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Table 12 1 Properties of DNA Polymerases

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Table 12-1. Properties of DNA PolymerasesE. coli I II III

Polymerization:

53

+ + +

Exonuclease activity:

35 + + +

53 +

Synthesis from:

Intact DNA Primed single

strands

+

Primed single

strands plus single-

strand-binding

protein

+ +

In vitro chain

elongation rate

(nucleotides per

minute)

600 ? 30,000

Molecules present

per cell

400 ? 1020

Mutation lethal? + +

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Mammalian Cells*

Polymerization:

53

+ + + + +

Exonuclease

proofreading

activity:3 5

+ + +

Synthesis from:

RNA primer + + ?

DNA primer + + + + +

Associated DNA

primase

+

Sensitive to

aphidicolin (inhibitor

of cell DNA

synthesis)

+ + +

Cell location:

Nuclei + + + +Mitochondria +

*Yeast DNA polymerase I, II, and III are equivalent to polymerase,, and, respectively. I and III are essential for cell viability.Polymerase is most active on DNA molecules with gaps of about 20 nucleotides and is thought to play a role in DNA repair.FEN1 is the eukaryotic 53exonuclease that removes RNA primers; it is similar in structure and function to the domain ofE.

colipolymerase I that contains the 53exonuclease activity.

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The first DNA polymerase was discovered by Arthur Kornberg in 1957: Pol I

E. coli DNA Pol I has 3 enzymatic activities:

1) Polymerization 5 32) Exonuclease 3 5 (Proofreading)3) Exonuclease 5 3 (Edit out sections of damaged DNA)

Klenow Fragment

DNA Polymerase Error Rate = 1/ 109 bp = 1 X 109 in the cell

100-1000X better than RNA Polymerase

DNA Pol III is highly processive while DNA Pol I is distributive

Processivity is continuous synthesis by polymerase without dissociation fromthe template.

A DNA polymerase that is Distributive will dissociate from the template aftereach nucleotide addition

Pol I & II main DNA repair enzymePol III main DNA replication enzyme

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Helicase -unwinds DNA. (ATP hydrolysis required -

introduces positive supercoils.)SSB protein (single-strand-binding protein) -binds to

the parental single strands as they are unwoundto prevent reannealing.

DNA gyrase -introduces negative supercoils to relievetorsional strain (ATP hydrolysis required).

RNA primase- (a specific RNA polymerase)synthesizes a primer of about 5bases long. TheRNA primer is later removed (and the gap filled

in) by Pol I.Pol III dimer-adds deoxyribonucleotides to the RNA

primer.

LEADING STRAND SYNTHESIS (elongation)

primosomeis now generally used to denote a complex between

primase and helicase, sometimes with other accessory proteins.

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Figure 5.11. Model of theE. colireplication fork

Model for the replication machine, or replisome

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Eukaryotic Replication Machinery Is Generally Similar to That ofE. coli

(refer to Table 2 for the proteins used)

TERMINATION OF DNA REPLICATION :-Pol I cleaves off RNA primers and fills in gaps (both

leading and lagging strands); as well as Rnase H(bacteria)

-DNA ligase seals gaps.

Figure 7-2. Plasmid DNA replication

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Telomerase Prevents Progressive Shortening of Lagging Strands duringEukaryotic DNA Replication

Termination of Eucaryotic DNA replication: The Problem - its a linearchromosome, so how to complete the ends?? (Cant just ligate ends and get a

circle as with E. coli chromosome;

Eucaryotic

Telomere structure

Telomerase

Ends of linear DNA will be

shortened by replication

Lagging strand cannot be primed beyond end of

leading strand, but the leading strand is

shortened due to priming.

Therefore, chromosomal end must be repaired

Telomerase is an RNA-directed DNApolymerase, containing RNA template.

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DNA replication leaves one incomplete end Telomere synthesis by telomerase

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Telomerase and Cancer

Germ cells and rapidly dividing somatic cells produce

telomerase.

Most human somatic cells lack telomerase, leading toshortening of telomeres with cell division.

Most tumor cells express telomerase.

Telomerase knockout mice are viable (!), but less able to

produce tumors.

Telomerase inhibitors may be valuablechemotherapeutics (e.g., Gerons GRN163L started

clinical trials for breast cancer August, 2008).

Telomerase activators may be valuable for regeneration

(e.g. Gerons TAT2 increases telomerase activity andproliferative capacity in cytotoxic T-cells in HIV-infected

pts.)

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DNA TOPOLOGY: DNA-BINDING PROTEINS ALTER THE TOPOLOGY OF DNA

Negative supercoiled circular DNA is compact and is energetically favored.

Most DNA in cells has negative supercoiled (right-handed)

superhelices.Superhelices are underwound. This facilitated DNA

helix unwinding for replication, recombination, transcription, etc.

Positive supercoils(left-handed) make opening the helix more difficult.

The topology of DNA (state of supercoiling) can be changed by unwinding or

winding supercoils. Changes in linking number result in different DNAtopoisomers. Changes require cutting one or both DNA strands.

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Different states of DNA supercoiling (negative and positive)

Topoisomerases,enzymes that catalyze the reversible breakage and

rejoining of DNA strands

Topoisomerase enzymes can DNA convert + to - supercoils

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Type I topoisomerases relax DNA (i.e., remove supercoils) by nicking and

closing one strand of duplex DNA

Topoisomerase I

[1 strand cut]

[left-handed supercoils]

Type II topoisomerases change DNA topology by breaking and rejoining

double-stranded DNA. These enzymes can introduce or remove supercoils and

can separate two DNA duplexes that are intertwined

Topoisomerase II[2 strands cut]

[right-handed supercoils]

(DNA Gyrase - uses ATP)

*Two DNA gyrase inhibitors arenalidixic acid (prevents strand

cutting and rejoining) and

novobiocin (blocks ATP binding) are.

Both replicated circular and linear DNA chromosomes are separatedby type II topoisomerases.

(NOTE: Helicase in DNA replication adds positive

supercoils, makes NO cuts, and uses ATP)

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The Role of Topoisomerases in DNA Replication

Figure 12-14. Action ofE. coli type Itopoisomerase (Topo I). The DNA-

enzyme intermediate contains acovalent bond between the 5-phosphoryl end of the nicked DNAand a tyrosine residue in the protein(inset). After the free 3-hydroxyl endof the red cut strand passes under theuncut strand, it attacks the DNA-enzyme phosphoester bond, rejoiningthe DNA strand. During each roundof nicking and resealing catalyzed byE. coli Topo I, one negative supercoilis removed. (The assignment of signto supercoils is by convention withthe helix stood on its end; in anegative supercoil the front strandfalls from right to left as it passes overthe back strand (as here); in a positive

supercoil, the front strand falls fromleft to right.)

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Figure 12-16. Action ofE. coli

DNA gyrase, a type IItopoisomerase. (a) Introduction

of negative supercoils. The initial

folding introduces no stable

change, but the subsequent

activity of gyrase produces a

stable structure with two

negative supercoils. Eukaryotic

Topo II enzymes cannot introduce

supercoils but can remove

negative supercoils from DNA. (b)

Catenation and decatenation oftwo different DNA duplexes. Both

prokaryotic and eukaryotic Topo

II enzymes can catalyze this

reaction.

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Fidelity of DNA replication can be traced to three distinct

activities:

1. accurate selection of nucleotides

2. immediate proofreading

3. postreplicative mismatch repair

DNA RepairTo maintain the integrity of their genomes,

cells have therefore had to evolve mechanisms to

repair damaged DNA.

A failure to repair DNA produces a mutation.

The recent publication of the human genome has already

revealed 130 genes whose products participate in DNA repair.

More will probably be identified soon.

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Agents that Damage DNA

Certain wavelengths of radiation

ionizing radiation such as gamma rays and x-rays

ultraviolet rays, especially the UV-C rays (~260 nm) that are

absorbed strongly by DNA but also the longer-wavelength UV-B that

penetrates the ozone shie ld

Highly-reactive oxygen radicals produced during normal cellular

respiration as well as by other biochemical pathways.

Chemicals in the environment

many hydrocarbons, including some found in cigarette smoke

some plant and microbial products, e.g. the aflatoxins produced

in moldy peanuts

Chemicals used in chemotherapy, especially chemotherapy of

cancers

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Figure 5.20. Examples of DNA

damage induced by radiation and

chemicals (A) UV light induces the

formation of pyrimidine dimers, inwhich two adjacent pyrimidines

(e.g., thymines) are joined by a

cyclobutane ring structure. (B)

Alkylation is the addition of methyl

or ethyl groups to various positions

on the DNA bases. In this example,alkylation of the O6 position of

guanine results in formation of O6-

methylguanine. (C) Many

carcinogens (e.g., benzo-(a)pyrene)

react with DNA bases, resulting in

the addition of large bulky chemicalgroups to the DNA molecule.

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Types of DNA Damage1.All four of the bases in DNA (A, T, C, G) can be covalently modified at various

positions.

One of the most frequent is the loss of an amino group ("deamination") resulting, for example, in a C being converted to a U.

2.Mismatches of the normal bases because of a failure of proofreading during

DNA replication.

Common example: incorporation of the pyrimidineU (normally found only in

RNA) instead ofT.

3.Breaks in the backbone.

Can be limited to one of the two strands (a single-stranded break, SSB) or

on both strands (a double-stranded break (DSB).

Ionizing radiation is a frequent cause, but some chemicals produce breaks as

well.

4.Crosslinks Covalent linkages can be formed between bases

on the same DNA strand ("intrastrand") or

on the opposite strand ("interstrand").Several chemotherapeutic drugs used against cancers crosslink DNA

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Figure 5.19. Spontaneous

damage to DNA There are twomajor forms of spontaneous

DNA damage: (A) deamination

of adenine, cytosine, and

guanine, and (B) depurination

(loss of purine bases) resulting

from cleavage of the bond

between the purine bases and

deoxyribose, leaving an

apurinic (AP) site in DNA.

dGMP = deoxyguanosine

monophosphate.

Table 12-2. DNA Lesions That Require Repair

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DNA Lesion Example/Cause

Missing base Removal of purines by acid and heat (underphysiological conditions 104 purines/day/cell in a

mammalian genome); removal of altered bases

(e.g., uracil) by DNA glycosylases

Altered base Ionizing radiation; alkylating agents (e.g.,

ethylmethane sulfonate)

Incorrect base Mutations affecting 35 exonuclease

proofreading of incorrectly incorporated bases

Bulge due to deletion or insertion of a nucleotide Intercalating agents (e.g., acridines) that cause

addition or loss of a nucleotide during

recombination or replication

Linked pyrimidines Cyclotubyl dimers (usually thymine dimers)

resulting from UV irradiation

Single- or double-strand breaks Breakage of phosphodiester bonds by ionizing

radiation or chemical agents (e.g., bleomycin)

Cross-linked strands Covalent linkage of two strands by bifunctional

alkylating agents (e.g., mitomycin C)

3-deoxyribose fragments Disruption of deoxyribose structure by free radicalsleading to strand breaks

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These mechanisms of DNA repair can be divided into two general

classes:

(1) direct reversal of the chemical reaction responsible for

DNA damage, and(2) Excision Repair- removal of the damaged bases followed

by their replacement with newly synthesized DNA.

Three types of excision repair

1. BASE-EXCISION REPAIR (BER)2. NUCLEOTIDE-EXCISION REPAIR,(NER)

3. MISMATCH REPAIR (MMR)

Postreplication Repair1. RECOMBINATIONAL REPAIR2. ERROR-PRONE REPAIR.

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Figure 5.21. Direct

repair of thymine

dimers UV-induced

thymine dimers can

be repaired by

photoreactivation, inwhich energy from

visible light is used to

split the bonds

forming the

cyclobutane ring.

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Figure 5.22. Repair of

O6-methylguanine

O6-methylguanine

methyltransferase

transfers the methyl

group from O6-

methylguanine to a

cysteine residue in

the enzyme's active

site.

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Figure 5-50. Acomparison of twomajor DNA repair

pathways.

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Figure 12-26. Excision repair of DNAbyE. coli UvrABC mechanism. Twomolecules of UvrA and one of UvrBform a complex that moves randomly

along DNA (steps 1 and 2). Once thecomplex encounters a lesion,conformational changes in DNA,powered by ATP hydrolysis, cause thehelix to become locally denatured andkinked by 130 (step 3). After the UvrA

dimer dissociates (step 4), the UvrCendonuclease binds and cuts thedamaged strand at two sites separatedby 12 or 13 bases (steps 5 and 6). UvrBand UvrC then dissociate, and helicaseII unwinds the damaged region (step

7), releasing the single-strandedfragment with the lesion, which isdegraded to mononucleotides. The gapis filled by DNA polymerase I, and theremaining nick is sealed by DNA ligase(step 8). [Adapted from A. Sancar andJ. Hearst, 1993, Science259:1415.]

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Figure 12-24. Model of mismatch

repair by the E. coliMutHLS system.

This repair system operates soon after

incorporation of a wrong base, before

the newly synthesized daughter strandbecomes methylated. MutH binds

specifically to a hemimethylated GATC

sequence, and MutS binds to the site

of a mismatch. Binding of MutL

protein simultaneously to MutS and to

a nearby MutH activates the

endonuclease activity of MutH, which

then cuts the unmethylated (daughter)

strand in the GATC sequence. A stretch

of the daughter strand containing the

mispaired base is excised, followed by

gap repair and ligation and then

methylation of the daughter strand.

[Adapted from R. Kolodner, 1996,

Genes and Develop.10:1433; see also

A. Sancar and J. Hearst, 1993, Science

259:1415.]

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Figure 5.25. Mismatch repair in E. coliFigure 5.26. Mismatch repair in

mammalian cells

T bl 5 1 E I l d i N l tid E i i R i

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Human Yeast Function

XPA RAD14 Damage recognition

XPB RAD25 Helicase

XPC RAD4 DNA binding

XPD RAD3 Helicase

XPF RAD1 5 nuclease

XPG RAD2 3 nuclease

ERCC1 RAD10 Dimer with XPF

Table 5.1. Enzymes Involved in Nucleotide-Excision Repair

Table 5-2. Inherited Syndromes with Defects in DNA Repair

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NAME PHENOTYPE ENZYME OR PROCESS AFFECTED

MSH2, 3, 6, MLH1, PMS2 colon cancer mismatch repair

Xeroderma pigmentosum (XP)

groups AG

skin cancer, cellular UV sensitivity,

neurological abnormalities

nucleotide excision-repair

XP variant cellular UV sensitivity translesion synthesis by DNA

polymerase

Ataxiatelangiectasia (AT) leukemia, lymphoma, cellular -ray sensitivity, genome instability

ATM protein, a protein kinaseactivated by double-strand breaks

BRCA-2 breast and ovarian cancer repair by homologous

recombination

Werner syndrome premature aging, cancer at several

sites, genome instability

accessory 3-exonuclease and DNA

helicase

Bloom syndrome cancer at several sites, stunted

growth, genome instability

accessory DNA helicase for

replication

Fanconi anemia groups AG congenital abnormalities,

leukemia, genome instability

DNA interstrand cross-link repair

46 BR patient hypersensitivity to DNA-damaging

agents, genome instability

DNA ligase I

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Figure 5.27. Postreplication repair The

presence of a thymine dimer blocks

replication, but DNA polymerase can bypassthe lesion and reinitiate replication at a

new site downstream of the dimer. The

result is a gap opposite the dimer in the

newly synthesized DNA strand. In

recombinational repair, this gap is filled by

recombination with the undamagedparental strand. Although this leaves a gap

in the previously intact parental strand, the

gap can be filled by the actions of

polymerase and ligase, using the intact

daughter strand as a template. Two intact

DNA molecules are thus formed, and the

remaining thymine dimer eventually can be

removed by excision repair

Repairing Strand Breaks

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-Ionizing radiation and certain chemicals can produce both:

1. single-strand breaks (SSBs) and

2. double-strand breaks (DSBs) in the DNA backbone.

Single-Strand Breaks (SSBs)

-breaks in a single strand of the DNA molecule are repaired using the same

enzyme systems that are used in Base-Excision Repair (BER).

Double-Strand Breaks (DSBs)-there are two mechanisms by which the cell attempts to repair a completebreak in a DNA molecule:

Direct joiningof the broken ends. This requires proteins that recognize and bind to

the exposed ends and bring them together for ligating. They would prefer to see

some complementary nucleotides but can proceed without them so this type ofjoining is also called Nonhomologous End-Joining (NHEJ).

A protein called Ku is essential for NHEJ. Ku is a heterodimer of the subunits Ku70

and Ku80. In the 9 August 2001 issue ofNature, Walker, J. R., et al, report the

three-dimensional structure of Ku attached to DNA. Their structure shows

beautifully how the protein aligns the broken ends of DNA for rejoining.

Figure 12-28. Repair of double-strand

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breaks by end-joining of

nonhomologous DNAs (dark and light

blue), that is, DNAs with dissimilar

sequences at their

ends. These DNAs could be cut

fragments from a single gene, or DNAs

cut from different chromosomes. A

complex of two proteins, Ku and DNA-

dependent protein kinase

, binds to the ends of a double-strand

break. After formation of a synapse in

which the broken ends overlap, Kuunwinds the ends, by chance revealing

short homologous sequences in the two

DNAs, which base-pair to yield a region

of microhomology. The unpaired single-

stranded 5 ends are removed by

mechanisms that are not well

understood, and the two double-stranded molecules ligated together. As a

result, the double-strand break is

repaired, but several base pairs at the

site of the break are removed. [Adapted

from G. Chu, 1997,J. Biol. Chem.

272:24097; M. Lieber et al., 1997, Curr.

Opin. Genet. Devel.7:99.]

E i di j i i b f h i l i h

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Errors in direct joining may be a cause of the various translocations that are

associated with cancers.

Examples:

Burkitt's lymphoma

the Philadelphia chromosome in chronic myelogenous leukemia (CML)B-cell leukemia

Homologous Recombination. Here the broken ends are repaired using the

information on the intactsister chromatid (available in G2 after chromosome duplication), or on the

homologous chromosome (in G1; that is, before each chromosome has been

duplicated). This requires searching around in the nucleus for the homolog a

task sufficiently uncertain that G1 cells usually prefer to mend their DSBs by

NHEJ. or on the

same chromosome if there are duplicate copies of the gene on the

chromosome oriented in opposite directions (head-to-head or back-to-back).

Two of the proteins used in homologous recombination are encoded by the genes

BRCA1 and BRCA2. Inherited mutations in these genes predispose women to breast

and ovarian cancers.

Homologous DNA

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BurkittLymphoma.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CML.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BCL-2.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Chromosomes.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CellCycle.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CellCycle.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CellCycle.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CellCycle.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CellCycle.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Chromosomes.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Chromosomes.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BCL-2.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BCL-2.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BCL-2.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CML.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CML.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CML.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/CML.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BurkittLymphoma.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BurkittLymphoma.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BurkittLymphoma.html

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Homologous DNA

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Figure 5-53. Two different

types of end-joining for

repairing double-strand

breaks. (A) Nonhomologous

end-joining alters the original

DNA sequence when

repairing broken

chromosomes. These

alterations can be either

deletions (as shown) or short

insertions. (B) Homologous

end-joining is more difficult

to accomplish, but is much

more precise.

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Inducible DNA-Repair Systems Are Error-Prone

-SOS repair system of bacteriathis system generates many errors in the DNA as it repairs lesions, it

is referred to asrepairs UV-induced damage, differs from the constitutive UvrABC

system

its activity is dependent on RecA protein

errors induced by the SOS system are at the site of lesions,

suggesting that the mechanism of repair is insertion of random

nucleotides in place of the damaged ones in the DNA.

*many investigators believe that in animal cells, as in bacteria,

most mutations are an indirect, not direct, consequence of DNA

damage.

Both bacterial and eukaryotic cells have inducible DNA-repair systems,

which are expressed when DNA damage is so extensive that replication

may occur before constitutive mechanisms can repair all the damage.

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Figure 8-4. Differenttypes of mutations

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Recombination- three different functions:

1. increasing genetic diversity which results in the exchange

of genes between paired homologous chromosomes

during meiosis

2.plays

also an important mechanism for repairingdamaged DNA

3. is involved in rearrangements of specific DNA sequences

that alter the expression and function of some genes

during development and differentiation

Thus, recombination plays important roles in the lives of individual cells and

organisms, as well as contributing to the genetic diversity of the species.

R bi ti

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Recombination

DNA rearrangements are caused by a set of mechanisms that

are collectively called genetic recombination.Two broad classes:

1. general recombination

2. site-specific recombination.

General recombination (also known as homologous recombination)-genetic exchange takes place between a pair of homologous DNAsequences

The breaking and rejoining of two homologous

DNA double helices creates two DNA

molecules that have crossed over. In meiosis,

this process causes each chromosome in a

germ cell to contain a mixture of maternally

and paternally inherited genes.

DNA Molecules Recombine by Breaking and Rejoining

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Figure 5.28. Models ofrecombination In copy

choice, recombination occurs

during the synthesis of

daughter DNA molecules.

DNA replication starts with

one parental DNA templateand then switches to a

second parental molecule,

resulting in the synthesis of

recombinant daughter DNAs

containing sequences

homologous to both parents.In breakage and rejoining,

recombination occurs as a

result of breakage and

crosswise rejoining of

parental DNA molecules.

DNA Molecules Recombine by Breaking and Rejoining

Holliday model

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Holliday model

Figure 5.31. The Holliday model

for homologous recombination

Holliday junction The centralintermediate in recombination,consisting of a crossed-strandstructure formed by

homologous base pairingbetween strands of two DNAmoleucles.

Figure 5 33 Isomerization and

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Figure 5.33. Isomerization and

resolution of Holliday

junctions Holliday junctions

are resolved by cutting and

rejoining of the crossed

strands. If the Holliday

junction formed by the initial

strand exchange is resolved,

the resulting progeny are

heteroduplexes but are not

recombinant for geneticmarkers outside of the

heteroduplex region. Two

rotations of the crossed-strand

molecule, however, produce

an isomer in which the

unbroken parental strands,rather than the initially nicked

strands, are crossed. Cutting

and rejoining of the crossed

strands of this isomer yield

progeny that are recombinant

heteroduplexes.

E I l d i H l R bi i

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Enzymes Involved in Homologous Recombination

1. RecA (aside from DNA polymerase, ligase and

single-stranded binding proteins)

central protein involved in homologous recombination promotes the exchange of strands between

homologous DNAs that causes heteroduplexes to form

capable of catalyzing, by itself, the strand exchange

reactions that are central to the formation of Holliday

junctions

action of RecA can be considered in three stages (see

next slide)

found in E. coli

2. RecBCD enzyme (most recombination events in E.coli) complex of 3 proteins (RecB, C and D).

initiates recombination by providing the single-stranded

DNA to which RecA binds by unwinding and nicking

double-stranded DNA .

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Figure 5.35. Function of the

RecA protein

1. RecA initially binds tosingle-stranded DNA to

form a protein-DNA

filament.

2. The RecA protein that

coats the single-stranded

DNA then binds to a

second, double-stranded

DNA molecule to form a

non-base-paired

complex.

3. Complementary basepairing and strand

exchange follow, forming

a heteroduplex region.

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Figure 5.36. Initiation of

recombination by RecBCD

1. The E. coliRecBCD

complex binds to the

end of a DNA molecule

and unwinds the DNA

as it travels along the

molecule.2. When it encounters a

specific sequence

(called a chi site*), it

nicks the DNA strand.

3. Continued unwindingthen forms a displaced

single strand to which

RecA can bind.

*specific nucleotide sequence

(GCTGGTGG)

3 R A B d C

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3. RuvA, B, and C

E. coliproteins become involved in recombination once

a Holliday junction is formed

Figure 5.37. Branch

migration and resolution of

Holliday junctions

1. Two E. coliproteins

(RuvA and RuvB)

together catalyze themovement of the

crossed-strand site in

Holliday junctions

(branch migration).

2. RuvC resolves theHolliday junctions by

cleaving the crossed

strands, which are then

joined by ligase.

RAD51

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RAD51

-RecA-related protein in yeast

-required for genetic recombination as well as for the repair

of double-strand breaks-able to catalyze strand exchange reactions in vitro

-Proteins related to RAD51 have been identified in complex

eukaryotes, including humans

*In yeasts:Holliday junctions are resolved by a complex of RAD1

and RAD10, with RAD1 cleaving single-stranded DNA at the

crossover junction. (RAD1 and RAD10 are homologs of the

mammalian XPF and ERCC1 DNA repair proteins and also

cleave damaged DNA during nucleotide-excision repair).