DNA REPLICATION-in all cells, DNA sequences should be maintained and replicated with high

fidelity (mutation rate, approximately 1 nucleotide change per 109 nucleotides each time the DNA is replicated, is roughly the same for organisms as different as bacteria and humans).

-the sequence of the human genome (approximately 3 × 109 nucleotide pairs) is changed by only about 3 nucleotides each time a cell divides.

-this allows most humans to pass accurate genetic instructions from one generation to the next, and also to avoid the changes in somatic cells 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

1) CancerCells are carefully controlled in the number of cell doublings that they are

capable of as well as when cell division will occur. In cancer the control of initiation of replication is lost2) Aging

For example, fibroblast cells (connective tissue) in culture will double for about 50 generations. Then they enter “senescence.” Senescent cells are no longer 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 the wrinkled skin of the elderly.3) DNA repair diseases

There are several diseases that cause premature aging or sensitivity to UV light.Examples include:a) Bloom Syndrome, a cancer-prone genetic disorder due to genetic instability in the form of increased frequencies of breaks of the chromosomes.b) Xeroderma Pigmentosum, a human DNA repair deficiency syndrome leading to predisposition to sun-light-induced skin cancer.c) Werner Syndrome, a premature aging disease that begins in adolescence or early adulthood and results in the appearance of old age by 30-40 years of age.

Xeroderma patient

KEY CONCEPTS:

• Proteins interact with DNA in all biological activities involving DNA.• 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 of eucaryotic 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 correction systems.

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

1958: Meselson and Stahl: DNA Replication is Semiconservative

*(in both prokaryotes & eukaryotes)

2. Most DNA Replication Is Bidirectional

Figure 12-2. Three mechanisms of DNA strand growth that are consistent with semiconservative replication. The third mechanism—bidirectional growth of both strands from a single origin—appears to be the most common in both eukaryotes and prokaryotes.

•Because of the anti-parallel structure of the DNA duplex, new DNA must be synthesized in both the 5’ to 3’ and 3’ to 5’ directions overall.•However all known DNA polymerases synthesize DNA in the 5’ to 3’ direction only.•The solution is semidiscontinuous DNA replication.

Leading Strand-replicates continuously

Lagging Strand-replicates discontinuously-consists of Okazaki Fragments (ss DNA chains 1000-2000 nucleotides long, primed by very short RNAprimers) which need to be joined by DNA ligase-the parental strand forms a “trombone structure”

RNA primers

The Leading and Lagging Strands Are Synthesized Concurrently

3. DNA Replication Begins at Specific Chromosomal Sites

-DNA synthesis is initiated at special regions called replication origins. A bacterial 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

Figure 5.14. Origin of replication in E. coli Replication initiates at a unique site on the E. coli chromosome, designated the origin (ori)

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

Replicon - region of DNA served by one replication origin.

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 by directing assembly of the replication machinery to specific sites on the DNA chromosome.

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

1. oriC is an ≈240-bp DNA segment present at the start site for replication of E. coli chromosomal DNA

-contain repetitive 9-bp and AT-rich 13-bp sequences, referred to as 9-mers (dnaA boxes) and 13-mers, respectively.

Figure 12-5. Consensus sequence of the minimal bacterial replication origin

*these are binding sites for the DnaA protein that initiates replication.

2. Yeast Autonomously Replicating Sequences- has multiple origins of replication-confers on a plasmid the ability to replicate in yeast and is a

required element in yeast artificial chromosomes -a 15-bp segment, designated element A, stretching from

position 114 through 128 which contains an 11-base-pair ARS consensus sequence (ACS), which is the specific binding site of the 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 yeast ARS element

3. SV40 Replication Origin-A 65-bp region in the SV40 chromosome is sufficient to promote DNA 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 binds to the origin and also acts as a helicase.

The DNA Replication MachineryDNA Polymerases

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

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

• The two strands in the DNA duplex are opposite (5 →3 and 3 →5 ) in ′ ′ ′ ′chemical polarity, but all DNA polymerases catalyze nucleotide addition at the 3 -′hydroxyl end of a growing chain, so strands can grow only in the 5 →3 direction.′ ′

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

E. coli protein Eukaryotic protein FunctionDnaA ORC proteins Recognition of origin of replication

Gyrase Topoisomerase I/II Relieves positive supercoils ahead of replication fork

DnaB Mcm DNA helicase that unwinds parental duplex

DnaC ? Loads helicase onto DNASSB RFA Maintains DNA in single-stranded state

γ-complex RFC Subunits of the DNA polymerase holoenzyme that load the clamp onto the DNA

pol III core pol δ/ε Primary replicating enzyme; synthesizes entire leading strand and Okazaki fragments; has proofreading capability

Β subunit PCNA Ring-shaped subunit of DNA polymerase holoenzzyme that clamps replicating polymerase to DNA;works with pol III in E. coli and pol δ or ε in eukaryotes

Primase Primase Synthesizes RNA primers- pol α Synthesizes short DNA oligonucleotides

as part of RNA-DNA primer

DNA ligase DNA ligase Seals Okazaki fragments into continuous strand

pol I FEN-1 Removes RNA primers; pol I of E. coli also fills gap with DNA.

Table 2. Some of the Proteins Required for Replication

Figure 12-7. Model of initiation of replication at E. coli oriC.

•DnaA Protein Initiates Replication in E. coli

•DnaB Is an E. coli Helicase That Melts Duplex DNA

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 fragments together

• All of this is fast, about 100 bp/second

Table 12-1. Properties of DNA Polymerases

E. coli I II III

Polymerization: 5′→3′

+ + +

Exonuclease activity:

3′→5′ + + +

5′→3′ + − −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 ? 10–20

Mutation lethal? + − +

Mammalian Cells* α β† γ δ ε

Polymerization: 5′→3′

+ + + + +

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 α, β, and δ, respectively. I and III are essential for cell viability.† Polymerase β 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 5′→3′ exonuclease that removes RNA primers; it is similar in structure and function to the domain of E. coli polymerase I that contains the 5′→3′ exonuclease activity.

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’ 3’2) 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 cell100-1000X better than RNA Polymerase

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

Processivity is continuous synthesis by polymerase without dissociation from the template.A DNA polymerase that is Distributive will dissociate from the template after each nucleotide addition

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

• Helicase -unwinds DNA. (ATP hydrolysis required - introduces positive supercoils.)• SSB protein (single-strand-binding protein) -binds to the parental single strands as they are unwound to prevent reannealing.• DNA gyrase -introduces negative supercoils to relieve torsional strain (ATP hydrolysis required).• RNA primase- (a specific RNA polymerase) synthesizes a primer of about 5bases long. The RNA 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)

primosome is now generally used to denote a complex between primase and helicase, sometimes with other accessory proteins.

Figure 5.11. Model of the E. coli replication fork

Model for the “replication machine,” or replisome

• Eukaryotic Replication Machinery Is Generally Similar to That of E. 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

•Telomerase Prevents Progressive Shortening of Lagging Strands during Eukaryotic DNA Replication

Termination of Eucaryotic DNA replication: The Problem - it’s a linear chromosome, so how to complete the ends?? (Can’t just ligate ends and get a circle as with E. coli chromosome;

• Eucaryotic“Telomere” structure

Telomerase• Ends of linear DNA will beshortened by replication• Lagging strand cannot be primed beyond end ofleading strand, but the leading strand isshortened due to priming.• Therefore, chromosomal end must be repaired• Telomerase is an RNA-directed DNApolymerase, containing RNA template.

DNA replication leaves one incomplete end Telomere synthesis by telomerase

Telomerase and Cancer• Germ cells and rapidly dividing somatic cells producetelomerase.• 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 toproduce tumors.• Telomerase inhibitors may be valuablechemotherapeutics (e.g., Geron’s GRN163L startedclinical trials for breast cancer August, 2008).• Telomerase activators may be valuable for regeneration(e.g. Geron’s TAT2 increases telomerase activity andproliferative capacity in cytotoxic T-cells in HIV-infectedpts.)

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

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

• Type I topoisomerases relax DNA (i.e., remove supercoils) by nicking and closing one strand of duplex DNATopoisomerase 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 are nalidixic acid (prevents strand cutting and rejoining) andnovobiocin (blocks ATP binding) are.

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

(NOTE: Helicase in DNA replication adds positivesupercoils, makes NO cuts, and uses ATP)

The Role of Topoisomerases in DNA Replication

Figure 12-14. Action of E. coli type I topoisomerase (Topo I). The DNA-enzyme intermediate contains a covalent bond between the 5 -phosphoryl end of ′the nicked DNA and a tyrosine residue in the protein (inset). After the free 3 -′hydroxyl end of the red cut strand passes under the uncut strand, it attacks the DNA-enzyme phosphoester bond, rejoining the DNA strand. During each round of nicking and resealing catalyzed by E. coli Topo I, one negative supercoil is removed. (The assignment of sign to supercoils is by convention with the helix stood on its end; in a negative supercoil the “front” strand falls from right to left as it passes over the back strand (as here); in a positive supercoil, the front strand falls from left to right.)

Figure 12-16. Action of E. coli DNA gyrase, a type II topoisomerase. (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 of two different DNA duplexes. Both prokaryotic and eukaryotic Topo II enzymes can catalyze this reaction.

Fidelity of DNA replication can be traced to three distinct activities:

1. accurate selection of nucleotides2. immediate proofreading3. 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.

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 ofcancers

Figure 5.21. Direct repair of thymine dimers UV-induced thymine dimers can be repaired by photoreactivation, in which energy from visible light is used to split the bonds forming the cyclobutane ring.

Figure 5.20. Examples of DNA damage induced by radiation and chemicals (A) UV light induces the formation of pyrimidine dimers, in which 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 chemical groups to the DNA molecule.

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 pyrimidine U (normally found only in RNA) instead of T.

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

Figure 5.19. Spontaneous damage to DNA There are two major 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.

DNA Lesion Example/Cause

Missing base Removal of purines by acid and heat (under physiological 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 3 →5 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 radicals leading to strand breaks

SOURCE: Adapted from A. Kornberg and T. Baker, 1992, DNA Replication, 2d ed., W. H. Freeman and Company, pp. 771–773.

Table 12-2. DNA Lesions That Require Repair

The 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 repair1. BASE-EXCISION REPAIR (BER)2. NUCLEOTIDE-EXCISION REPAIR,(NER)3. MISMATCH REPAIR (MMR)

Postreplication Repair1. RECOMBINATIONAL REPAIR 2. ERROR-PRONE REPAIR.

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.

Figure 5-50. A comparison of two major DNA repair pathways.

Figure 12-26. Excision repair of DNA by E. coli UvrABC mechanism. Two molecules of UvrA and one of UvrB form a complex that moves randomly along DNA (steps 1 and 2). Once the complex encounters a lesion, conformational changes in DNA, powered by ATP hydrolysis, cause the helix to become locally denatured and kinked by 130° (step 3). After the UvrA dimer dissociates (step 4), the UvrC endonuclease binds and cuts the damaged strand at two sites separated by 12 or 13 bases (steps 5 and 6). UvrB and UvrC then dissociate, and helicase II unwinds the damaged region (step 7), releasing the single-stranded fragment with the lesion, which is degraded to mononucleotides. The gap is filled by DNA polymerase I, and the remaining nick is sealed by DNA ligase (step 8). [Adapted from A. Sancar and J. Hearst, 1993, Science 259:1415.]

Figure 12-24. Model of mismatch repair by the E. coli MutHLS system. This repair system operates soon after incorporation of a wrong base, before the newly synthesized daughter strand becomes 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.]

Figure 5.25. Mismatch repair in E. coli Figure 5.26. Mismatch repair in mammalian cells

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

NAME PHENOTYPE ENZYME OR PROCESS AFFECTED

MSH2, 3, 6, MLH1, PMS2 colon cancer mismatch repairXeroderma pigmentosum (XP) groups A–G

skin cancer, cellular UV sensitivity, neurological abnormalities

nucleotide excision-repair

XP variant cellular UV sensitivity translesion synthesis by DNA polymerase δ

Ataxia–telangiectasia (AT) leukemia, lymphoma, cellular γ-ray sensitivity, genome instability

ATM protein, a protein kinase activated 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 A–G congenital abnormalities, leukemia, genome instability

DNA interstrand cross-link repair

46 BR patient hypersensitivity to DNA-damaging agents, genome instability

DNA ligase I

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

Figure 5.27. Postreplication repair The presence of a thymine dimer blocks replication, but DNA polymerase can bypass the 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 undamaged parental 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

-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 complete break in a DNA molecule:

•Direct joining of 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 of joining 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 of Nature, 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 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, Ku unwinds 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.]

•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 intact

sister 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

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.

Inducible DNA-Repair Systems Are Error-Prone-SOS repair system of bacteria• this system generates many errors in the DNA as it repairs lesions • repairs 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.

Figure 8-4. Different types of mutations

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 repairing

damaged DNA3. 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.

Recombination•DNA rearrangements are caused by a set of mechanisms that are collectively called genetic recombination.

• Two broad classes:1. general recombination2. site-specific recombination.

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

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.

Figure 5.28. Models of recombination In copy choice, recombination occurs during the synthesis of daughter DNA molecules. DNA replication starts with one parental DNA template and 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

Figure 5.31. The Holliday model for homologous recombination

Holliday junction The central intermediate in recombination, consisting of a crossed-strand structure formed by homologous base pairing between strands of two DNA moleucles.

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 genetic markers 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.

Enzymes Involved in Homologous Recombination1. 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 .

Figure 5.35. Function of the RecA protein

1. RecA initially binds to single-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 base pairing and strand exchange follow, forming a heteroduplex region.

Figure 5.36. Initiation of recombination by RecBCD

1. The E. coli RecBCD 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 unwinding then forms a displaced single strand to which RecA can bind.

*specific nucleotide sequence (GCTGGTGG)

3. RuvA, B, and C• E. coli proteins become involved in recombination once

a Holliday junction is formed

Figure 5.37. Branch migration and resolution of Holliday junctions1. Two E. coli proteins

(RuvA and RuvB) together catalyze the movement of the crossed-strand site in Holliday junctions (branch migration).

2. RuvC resolves the Holliday junctions by cleaving the crossed strands, which are then joined by ligase.

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).