Chapter 30: DNA Replication, Repair, and Recombination

Chapter 30: DNA Replication, Repair, and Recombination

1. DNA Replication: An overview 2. Enzymes of Replication 3. Prokaryotic Replication 4. Eukaryotic Replication 5. Repair of DNA 6. Recombination and Mobile Genetic Elements 7. DNA Methylation and Trinucleotide Repeat Expansion

DNA Replication DNA double strand -> template for duplication, Replication Chemically similar to transcription As complex as translation but enzymes in only few copies/cell Extremely accurate: 10-10 mistakes/base Extremely regulated: only once per cell division

Action of DNA polymerase Template dntps 5 -> 3 direction Semi-conservative

Replication of DNA Unwinding of dsdna: - Rate in E. coli: 1000nt/sec - 100rev/sec (10bp/turn) Negative supercoils by: DNA Gyrase type II topoisomerase, ATP

E. coli theta replication autoradiogramm branch point called replication fork unidirectional / bidirectional prokaryotes and bacteriophages have only one origin of replication

Unidirectional vs. bidirectional θ replication [3H]tymidine pulse-labelling

Semidiscontinuous DNA replication Discontinous! Okazaki fragments: 1000-2000nt in prokaryotes 100-200nt in eukaryotes Joined by DNA ligase

Replication eye in Drosophila melanogaster DNA

Priming of DNA synthesis by short RNA segments E. coli: RNA Polymerase Primase, rifampicin sensitive Removal of RNA primers

2. Enzymes of replication DNA Replication requires (in order of appearance): 1. DNA Topoisomerase 2. Helicases 3. ssdna binding proteins 4. RNA primer synthesis 5. DNA polymerase 6. Enzyme to remove RNA primers 7. Link Okazaki fragments

E. coli DNA polymerase I in complex with a dsdna Arthur Kornberg, 1957 DNA Polymerase I 5 ->3 synthesis Processive, 20nt Recognizes dntp based on base pairing Right hand sructure Editing activity: 3 ->5 exonuclease 5 ->3 exonuclease (proofreading) Fidelity 10-7 Klenow fragment Lacks 5 ->3 exo, lacks N- term.

Nick translation as catalyzed by Pol I Used to radiolabel DNA probes for Southern/Northern DNaseI, αp 32 dntp

Pol I functions to repair DNA E. coli, Pol I mutant are viable but sensitive to UV and chemical mutagens Essentisl physiological function of Pol I 5 ->3 exonuclease is to excise RNA primers, role in replication

DNA Polymerase III Pol III is replicase of E. coli Holoenzyme consists of more than 10 subunits β subunit confers processivity >5000nt β subunit form a ring like sliding clamp with 80Å diameter hole, sliding clamp/ β clamp

Properties of E. coli DNA Polymerases

Components of E. coli DNA Polymerase III Holoenzyme

β subunit of E. coli Pol III holoenzyme

Unwinding of DNA 3 proteins required to advance replication fork: Helicase, DnaB, hexameric, ATP-dep., 5 ->3,AAA+ Strand separation, Rep helicase, dimer, ATP-dep. ssdna binding protein, prevent re-annealing, tetramer

Unwinding and Binding Proteins of E. coli DNA Replication

Active, rolling mechanism for DNA unwinding by Rep helicase

DNA ligase Ligating single strand nicks between Okazaki fragments E. coli: NAD-dependent T4 phage, ATP-dependent blunt end ligation

Primase Synthesis of RNA primers fro Okazaki fragments: 5 ->3 In vitro 11nt ±1

Prokaryotic Replication Bacteriophages Coliphages: M13, φx174 M13: 6408nt ssdna(+), circular Replication->RF Leading strand synthesis

φx174 Replication 5386nt ssdna circular Replication more complex than M13 Requires primosome Paradigm for lagging strand synthesis 6step process a. coating b. primosome assembly c. migration d. priming e. Pol III extension f. Pol I removes primers g. ligation, supercoiling

Micrograph of a primosome

Proteins of the Primosome a

The rolling circle mode of DNA replication a. Specific cut at + strand b. Extension of + strand c. Tandem-linked + strands d. Separation by endonuclease e. packaging Rolling circle = Sigma replication

φx174 (+) strand replication by the looped rolling circle mode φx174 (+) strand synthesis as model for leading strand replication 1. Cut by gene A protein 2. Pol II extension 3. Cut + ligation

The replication of E. coli DNA Bidirectional, theta replication leading and lagging strand synthesis occurs on a common 900kD multisubunit particle: the replisome -> loop of lagging strand Initiation: at oric, 245bp segment

The replication of E. coli DNA

A model for DNA replication initiation at oric oric, 245bp segment Contains 5 DnaA boxes Melting, P1 Penicillium citrinum endodunclease Specific for ssdna Prepriming complex (DnaB DnaC)6

Initiation of DNA replication is strictly regulated Only 1 replication/cell cycle Doubling time 20min-10h 1000nt/sec 4.6 10 6 bp genome -> 40min/replication -> multiforked chromosomes Sequestration of hemimethylated oric

Electron micrograph of an intact and supercoiled E. coli chromosome attached to two fragments of the cell membrane

Schematic diagram of the clamp loading cycle β clamp responsible for high processivity of Pol III Must be loaded onto DNA by a clamp loader ATP-dep. AAA+

Termination of replication Large 350 kb region in E. coli genome Flanked by 7 nonpalindromic nearly identical termination Sites Replication fork counterclockwise passes through TerG,F, B, and C but stops at TerA Analogous for other direction Ter act as valves Ter-action requires binding of Tus protein Without Ter, collision of replication forks terminates

Fidelity of Replication Complexity of replication (>20 proteins) important for high fidelity: T4 phage reversion 10-8 - 10-10 High accuracy due to: 1. Balanced dntp levels 2. Polymerase reaction itself, pairing 3. 3 ->5 exonuclease of Pol I and Pol III 4. Repair systems -> see later

Why only 5 ->3 synthesis? 3 ->5 extension would require retention of 5 triphosphate This would be lost upon editing!

Eukaryotic Replication Remarkable degree of similarity to prok. replication But linear chromosomes -> ends? Cell cycle regulation, can last 8h to > 100 days Most variation in G1 phase/go phase Irreversible decision to proliferate is made in G1 Checkpoint Controlled by cyclins and cyclin-dep. kinases Best understood from yeast (budding, fission)

The eukaryotic cell cycle

Eukaryotic cells contain many polymerases 6 families: A, E. coli Pol I, Pol γ (mitochondrial) B, E. coli Pol II, Pol α, Pol δ C, E. coli Pol III D, X, Y Pol δ, unlimited processivity when in complex with PCNA, proliferating cell nuclear antigen (systemic lupus erythematosus), β clamp function

Properties of Some Animal DNA Polymerases

Structure of PCNA

Eukaryotic chromosomes consist of numerous replicons Multiple replication origins, every 3-300kb Replication rate 50nt/sec, 20x slower than E. coli But 60x more DNA Replication would require 1 month Clusters of 20-80 adjacent replicons Not simultaneously, but ensure they initiate only once

Assembly of the initiator complex in 2 stages To prevent multiple rounds of initiation: Assembly of pre-rc in G1 phase (licensed) Activation at S phase Origin can fire only once Origin = ARS (autonomously replicating sequences) Re-replication prevented by Cdks and Geminin ORC, origin recognition complex Hexamer, Orc1-Orc6 (DnaA analog) MCM, minichr. maintenance funct.

Removal of RNA primers 2 enzymes: RNase H1, removes most of the RNA leaving a single 5 ribonucleotide (H, hybrid) Flap endonuclease-1 (FEN1) removes single single 5 ribonucleotide

Mitochondrial DNA is replicated in D- loops 15kb circular genome Leading strand synthesis precedes lagging strand Leading strand forms displacement loop (D-loop)

Reverse transcriptase Retroviruses: RNA containing eukaryotic viruses, e.g. HIV Replicate from RNA genome Copy RNA into DNA by Reverse Transcripase (RT) Similar to Pol I, 5 ->3 synthesis of DNA from RNA template, primed by host trna RNA is degraded by RNase H ssdna directs dsdna synthesis dsdna integration into host genome RT: important tool for cdna synthesis, oligo-dt primed

Reverse transcriptase

Structure of HIV-1 reverse transcriptase

RT inhibitors

Telomers and Telomerase How are the ends of linear chromosomes replicated? Problem: no priming at 5 of lagging strand possible without shortening of the chromosome upon every replication Telomer sequence: unusual, G-rich, 3 overhang (20-200bp) Specialized enzyme: telomerase adds G-rich repeats without teplate, is ribonucleoprotein, RNA acts as template

Synthesis of telomeric DNA by Tetrahymena telomerase

Telomers must be capped Without telomerase, chromosome would shorten 50-100nt with every cell division Exposed telomeric ssdna must be protected by capping with proteins, Pot1

Telomere length correlates with aging Primary cells in culture die after 20-60 divisions Such somatic cells have no telomerase activity -> Telomers shorten with every division Telomerase is active only in germ cells Analysis of fibroblast from donors of different age: No correlation with numbers of doublings in culture But correlation of telomer length with numbers of doublings Progeria: premature aging disease patients have short telomers

Cancer cells have active telomerase Why do somatic cells down regulate telomerase? Senescence may be a mechanism to protect from cancer All immortal cells express telomerase

Telomeric DNA can dimerize via G-quartets

Telomers form T-loops

Repair of DNA DNA is not inert UV radiation, ionizing radiation, toxic chemicals, oxidative metabolism can harm DNA Spontaneous hydrolysis of 10 000 glycosidic bonds in every cell every day... Human genome 130 genes dedicated to DNA repair Chemically similar in E. coli

Chemical damage of DNA Oxidation Hydrolysis Methylation

Direct reversal of damage Pyrimidine dimers are split by photolyase: UV (200-300nm) promtes Formation of cyclobutyl ring between adjacent thymine -> intrastrand thymine dimer

DNA photolyase Photoreactive enzyme: Absorbed light is transferred to FADH - Electron used to split thymine dimer Base flipping: Often used to repair damaged DNA

Excision repair Cells have two types of excision repair: 1. Nucleotide excision repair, NER repairs bulky lesions 2. Base excision repair, BER repairs nonbulky lesions involving a single base

Excision repair (NER) Found in all cells Activated by helix distortion Major defense in humans (cigarette smoke, carcinogens) 16 subunits, 3 in bacteria E. coli: UvrA, UvrB, UvrC UvrABC endonuclease 1. Cleavage 2. Displacement, UvrD 3. Repair, Pol I

NER diseases Xenoderma pigmentosum skin cells cannot repair UV damage Individuals extremely sensitive to sun light skin tumors risk 2000-fold elevated cultured skin cells are defective in repairing tymidine dimers Cell fusion experiments: 8 complementation groups Cockayne syndrome light sensitive and neurological defects demyelination-> oxidative damage in neurons

Base excision repair Single base repair: 1. DNA glycosidase-> Apurinic or apyrimidinic (AP) site (abasic site) 2. Ribose cleaved by AP endonuclease 3. Exonuclease 4. Filled by pol and ligase

Uracil in DNA would be highly mutagenic Why use thymine in DNA and uracil in RNA? Cytosine deaminates to uracil If U in DNA: no way to discriminate whether G-U mismatch is due to: G-C -> deaminated to U A-U Since T is normal in DNA, every U is due to deaminated C

Mismatch repair Replicational mispairing is repaired by mismatch repair (MMR) Defects result in hereditary nonpolyposis colorectal cancer (HNPCC) Must distinguish between correct and wrong base In E. coli, possible due to hemimethylation 3 proteins, MutS, MutL, MutH

Mismatch repair in E. coli 1. MutS binds mismatch as dimer 2. MutS-DNA recrutes MutL 3. MutS-MutL scan DNA for hemi- Methylated GATC, recrute MutH 4. Cleavage of non-methylated strand 5. Strand separation by UvrD 6. Exonuclease 7. Fill Pol III 8. Ligate

The SOS response On heavy DNA damage, E. coli stops to grow and induces DNA repair system, SOS system SOS operon, reca, uvra, uvrb repressed by LexA RecA is ssdna binding protein, induces cleavage of LexA upon ssdna binding -> release repression of SOS operon

Regulation of the SOS response in E. coli

SOS repair is error prone If replisome encounters DNA lesion: Stallment, relase Pol III core, collapse of replication fork To resume: either SOS repair or recombination repair Recombination repair: circumvents lesion and uses homologous recombination to restore damaged site (->later) In SOS repair, Pol III is replaced by bypass DNA polymerase, Pol IV or Pol V Error prone polymerases -> SOS response is mutagenic -> Adaptation to difficult situation by generating diversity

Double-strand break repair Ionizing radiation and free radicals can induce double strand breaks in DNA (DSB) Also induced by some cellular processes, e.g. VDJ recomb. 2 ways to repair DSBs: 1. Recombination repair-> later 2. Nonhomologous end-joining (NHEJ) involves DNA end binding protein Ku

Nonhomologous end-joining (NHEJ)

Identification of carcinogens Many forms of cancers are caused by exposure to certain chemical agents, carcinogens (man-made or natural) Ames test assay for carcinogenicity Salmonelle typhimurium his - incubate with chemical -> rate of reversion to his + correlates with mutagenecity of tested chemical

The Ames test for mutagenesis Filter disc containing Substance: 1. Zone lethal 2. Zone mutagenic 3. Zone spontaneous reversion

Recombination and mobile genetic elements Pairs of allelic genes may exchange chromosomal location by genetic recombination via homologous recombination Homologous recombination: Exchange of homologous segments between two DNA molecules Bacteria, haploid, exchange via conjugation (mating) or Transduction (viral)

The Holliday model of homologous recombination 1. ssdna nick 2. Strand invasion 3. Branch migration 4. Holliday interm. Chi structure 5. Resolution

Homologous recombination between two circular DNA duplexes Results either in two circles of the original sizes or in a single composite circle

Homologous recombination in E. coli is catalyzed by RecA RecA mutants have 10 4 -fold lowe rate of recombination RecA catalyzes ATP-dependent strand exchange Binds DNA with 6.2 RecA monomers/turn

Electron microscopy based image (of an E. coli RecA dsdna ATP filament

Model for RecA-mediated pairing and strand exchange

RecA-catalyzed assimilation of a single-stranded circle Requires: -free end (nick) -homology at 5

Hypothetical model for the RecAmediated strand exchange reaction Rad51 is eukaryotic homologue of RecA

recbcd initiate recombination by making single-strand nicks Products of the SOS operon Unwinding dsdna exonuclease to Chi sequence GCTGGTGG Every 5kb Have elevated rate of recombination Requires free ds ends: Transformation Conjugation, Transduction Replication fork collaps

RuvABC mediates branch migration and the resolution of the Holliday junction Branch migration is ATP-dependent, unidirectional Mediated by SOS-induced proteins: RuvB, ATP-dep. Pump, hexamer, AAA+ RuvA, binds Holliday junction, homotetramer RuvC, exonuclease

Recombination repair Transformation, transduction and conjugation are rare events requiring recombination Frequent is collapse of replication fork, 10times/euk cell cycle -> Recombination Repair 1. Replication arrest at lesion 2. Fork regression, chicken foot 3. Fill by Pol I 4. Reverse branch migration (RecG) 5. Replication restart Note: lesion is not repaired

Recombination repair of a singlestrand nick Replication fork encounters ss nick: 1. Collapse 2. RecBCD + RecA invasion 3. Branch migration, RuvAB 4. Resolution, RuvC -> nick has become 5 end of Okazaki fragment

Recombination repair reconstitutes doulbe-strand breaks Homologous end-joining as alternative to NHEJ 2 Holliday junctions intermediate 1. Resection of DS ends 2. DNA dynthesis and ligation 3. Resolution of 2 Hol.j.

Transposition and sitespecific recombination 1950 Barbara McClintock, varied pigmentation on maize Due to the action of variable genetic elements, i.e. non-mendelian inheritance 20 years later, evidence for mobile genetic elements in E. coli Transposable elements, transposons in prokaryotes and euk. Each transposon encodes for a transposase that catalyzes illegitimate recombination, because it requires no homology between donor and acceptor Transposition is mutagenic and dangerous, tightly regulated: 10-5 to 10-7 events per cell division

Prokaryotic transposons 3 Types: 1. Simplest, insertion sequences, IS Elements <2000bp, transposase, flanked by short inverted repeats, flanked by direct repeat at insertion site, E. coli: 8 copies of IS1, 5 copies of IS2

Properties of Some Insertion Elements

Transposons (2) 3 Types: 2. More complex, carry additional genes, e.g. antibiotic resistance Example, Tn3, 4957 bp a. transposase, TnpA b. Recombinase, TnpR c. beta-lactamase, Ampicilin resistance

Transposons (3) 3 Types: 3. Composite transposons gene containing central region flanked by IS-like modules that have the same or inverted orientation

Generation of direct repeats of the target sequence by transposon insertion

Two modes for transposition 1. Direct or simple transposition -> transposon moves from position A to position B 2. Replicative transposition -> transposon remains + new copy at position B

Direct transposition of Tn5 by a cut and paste mechanism 1. Transposase binding 2. Dimerization 3. Synaptic complex 4. Target capture 5. Integration

Replicative transposition

A cointegrate

Model for transposition via cointegrate 1. Pair of staggered ss cuts 2. Ligation of both ends at integration site forms replication fork 3. Replication forms cointegrate 4. Site-specific recombination cointegrate resolved

γδ Resolvase catalyzed sitespecific recombination Via double-strand DNA cleavage

Replicative transposons are responsible for much genetic remodeling in prokaryotes Transposons induce rearrangements in host genome a) Inversion of genomic segment b) Deletion of genomic segment Mediate transfer of genetic material between species

Phase variation is mediated by site-specific Recombination Salmonella typhimurium make 2 antigenetically distinct versions of flagellin, H1 and H2 only one of the two is expressed switch every 1000 cell generations, phase variation may help evade host immune response H2 is linked to rh1, that encodes a repressor for H1 Expression of H2-rh1 unit is controlled by a 995bp segment that contains 1. Promoter for H2-rh1 2. Hin gene coding for Hin DNA invertase 3. Two closely related 26bp sites, hixl and hixr

Mechanism of phase variation in Salmonella

Cre-mediated site-specific recombination Many bacteriophages have two modes to propagate: 1.lytic, lysis of cells 2. Lysogeic, integration into host genome Examples: Bacteriophage lambda, λ integrase P1 bacteriophage, Cre recombinase

The circularization of linear bacteriophage P1 DNA 34bp LoxP site, palindromic except for central 8bp

Mechanism of Cre loxp sitespecific recombination Via 3 -PhosphoTyr intermediate

Structure of the Cre tetramer complexed with loxp DNA

Most transposition in eukaryotes involve RNA intermediates 3% of the human genome consists of transposons Many are fosils, i.e. sequence mutated to be inactive Many ressemble retroviruses in sequence Retroposons Transposition via RNA intermediate, tanscription dsdna via reverse transcriptase, cdna Random integration by integrase Retroviral genome flanked by LTR, long terminal repeats (250-600bp) 3 polyproteins: gag (viral core) pol (reverse transcriptase) env (viral envelope)

Organization of retroviruses and the Ty1 retrotransposon

Non-viral retroposons Vertebrate genomes contain Retroposons that lack LTRs Non-viral retroposons,e.g. LINEs, long intersoersed nuclear elements, 1-7kn long Contain 2 ORFs ORF1, similar to gag ORF2, similar to pol In humans, LINEs account for 20% of genome!

DNA methylation and trinucleotide repeat expansion Species specific methylation of A and C residues in DNA to: N 6 -methyladenine (m 6 A) N 4 -methylcytosine (m 4 C) 5-methylcytosine (m 5 C)

DNA methylation Bacterial DNA is methylated at own restriction site E.coli, Dam methyltransferase (dam MTase), A in GATC Dcm MTase bith C in CCA/TGG at pos 5 both palindromic, mismatch repair and oric Methyl groups project into major groove of B-DNA, interact with DNA-binding proteins

The MTase reaction occurs via a covalent intermediate in which the target base is flipped out Methylation uses SAM, S-adenosylmethionine as methyl donor via a Cys thiolate attack, uses base flipping Inhibited by 5-fluorocytosine

Base flipping

DNA methylation in eukaryotes functions in gene regulation 5-methylcytosine is the only methylated base in most eukaryotes Modification in largely in GC dinucleotide CG is present at 1/5 of statistical expectation Upstream regions of many genes have CpG island

DNA methylation in eukaryotes Experimental assessment: Comparative southern blot of DANN cut with HpaII, cleaves CCGG, but not C-m 5 C-GG and MspI, cleaves both Identification of m 5 C residues through bisulfite sequencing DNA is reacted with bisulfite (HSO 3- ) which deaminates C to U, but not m 5 C, followed by PCR amplification: copies U to T and m 5 C to C Sequence and compare to untreated

DNA methylation in eukaryotes (2) Methylation switches off eukaryotic gene expression, particularly when methylation occurs in promoter region For example, globin genes are less methylated in erythroid cells Recognized by methyl-cpg binding domain (MBD) May also affect chromatin packaging

DNA methylation in eukaryotes is selfperpetuating Maintenance of methylation after replication -> inherited, Epigenetic inheritance: Non-Mendelian inherited information By DNMT1, which has preference for hemimethylated sites DNMT1 null mice die early in embr. devel.

Methylation is dynamic Pattern of DNA methylation varies in early embryological development: Methylation levels high in gamets (sperm, ova) but nearly eliminated in blastocyst stage Methylation then rises again till gastrula stage when it reaches that found in adults, remain constant Except germ line cells, remain unmethylated Pattern of expression differs in embryonic and somatic cells => Explains high failure of cloning experiments, few survivers, early death, abnormalities, large size

Genomic imprinting results from differential DNA methylation Difference in maternal and paternal inheritance: Mare x Male donkey -> mule Female donkey x stallion -> hinny Both are sterile mule hinny Maternal and paternal genes are differentially expressed = genomic imprinting, only in mammals No embry from transplant of two male or female pronuclei

DNA methylation is associated with cancer Most prevalent mutation is is m 5 C to T, covert proto-oncogens to oncogens or inactivate tumor suppressors

Several neurological diseases are associated with trinucleotide repeat expansion Fragile X syndrome: mental retardation, long narrow face 1 in 4500 males, 1 in 9000 females Activated by passage through female Affects FMR1 gene, which contains (CGG)n, n=6-60 in 5 region, n can increase from 60 to 200 = premutation Can the expand upon transmission to a daughter to >200 = full mutation Expansion arises through slippage during replication FMR1 is unmethylated in normal individuals But is methylated when premutation is maternally transmitted

Other important trinucleotide repeat diseases Huntington s disease (HD), 1 in 10 000, onset at age of approx. 40, 18-year course, fatal Protein huntingtin contains (CAG)n repeats (Gln) Normal 11-34, sick 37-86 Repeat length is unstable, changes in >80% meiotic transmissions Number of repeats inversely correlates with age of onset polygln aggregates as β sheets Neurons contain inclusions

The loop-out mechanism for the alteration of the number of consecutive triplet repeats in DNA through its replication