Replication Maintain genetic info from generation to generation Rapid (before cell divides) Accurate Need to correct replication errors and repair damage Recombination: shuffle pieces of DNA DNA Replication, Repair and Recombination Chapter 20 -

Each strand of DNA acts as a template for synthesis of a new strandEach strand of DNA acts as a template for synthesis of a new strand Daughter DNA contains one parental and one newly synthesized strandDaughter DNA contains one parental and one newly synthesized strand Semiconservative DNA replication

Replication involves initiation, elongation, and termination.Replication involves initiation, elongation, and termination. E. coli chromosome is circular, double-stranded DNAE. coli chromosome is circular, double-stranded DNA (4.6x10 3 kilobase pairs, >1000 bp/sec) Replication begins at a unique site (origin)Replication begins at a unique site (origin) Proceeds bidirectionally until the two replication complexes meet (termination site)Proceeds bidirectionally until the two replication complexes meet (termination site) Replisome - protein machinery for replication (one replisome at each of 2 replication forks)Replisome - protein machinery for replication (one replisome at each of 2 replication forks) Chromosomal DNA Replication is Bidirectional

Eukaryotic replication Eukaryotic chromosomes are large and linear DNA moleculesEukaryotic chromosomes are large and linear DNA molecules Fruit fly large chromosomes ~5.0x10 4 kb (~10x larger than E. coli) Multiple sites of initiation of DNA synthesisMultiple sites of initiation of DNA synthesis (versus one site in E. coli) Thus, overall time frame to complete replication is the same as in E coli

Drosophila DNA replicating Large # of replication forks at opposite ends of “bubbles” of duplicated DNA

Drosophila DNA replicating Rate of fork movement is slower (chromatin structure) Larger genome size Multiple sites of initiation Thus, overall time frame to complete replication is the same as in E coli

E. coli contains three DNA polymerases DNA polymerase I - repairs DNA and participates in DNA synthesis of the lagging strandDNA polymerase I - repairs DNA and participates in DNA synthesis of the lagging strand DNA polymerase II - role in DNA repairDNA polymerase II - role in DNA repair DNA polymerase III - the major DNA replication enzyme, responsible for chain elongationDNA polymerase III - the major DNA replication enzyme, responsible for chain elongation , ,  = polymerization core  4 = two sliding clamps.  complex = assembly. DNA Polymerase DNA polymerase III DNA directed DNA polymerase III 10 diff subunits

DNA Polymerase Synthesizes Two Strands Simultaneously DNA polymerase III

DNA polymerase III is a processive (rather than distributive) enzyme (remains bound to the replication fork until replication is complete)DNA polymerase III is a processive (rather than distributive) enzyme (remains bound to the replication fork until replication is complete)  -Subunits forms a sliding clamp which surrounds the DNA molecule  -Subunits forms a sliding clamp which surrounds the DNA molecule DNA Polymerase III Remains Bound to the Replication Fork Few Pol III needed for complete replication Allows rapid rate

DNA polymerase III is a processive (rather than distributive) enzyme (remains bound to the replication fork until replication is complete)DNA polymerase III is a processive (rather than distributive) enzyme (remains bound to the replication fork until replication is complete)  -Subunits forms a sliding clamp which surrounds the DNA molecule  -Subunits forms a sliding clamp which surrounds the DNA molecule DNA Polymerase III Remains Bound to the Replication Fork Few Pol III needed for Complete replication Allows rapid rate Similar concept used in other systems Bacteriophage pol

Chain Elongation Is a Nucleotidyl-Group-Transfer Reaction One nucleotide at a time dNTP substrate forms base pair 3’OH nucleophilic attack onto alpha phosphate of dNTP PPi cleavage…..does what??? 5’ to 3’ direction Requires template and primer to syn DNA (3’OH)

Proofreading Corrects Polymerization Errors DNA polymerase III holoenzyme also possesses 3’ 5’ exonuclease activity removes mispaired nucleotide before polymerization continues Recognizes distortion in the DNA caused by incorrectly paired bases Pol III can catalyze both chain elongation and degradation Polymerase error = 10 -5, nuclease error ; Overall error rate = Lowest rate of any enzyme but mistakes do occur and transmitted

DNA Polymerase Synthesizes Two Strands Simultaneously DNA pol III catalyzes chain elongation only in the 5’3’ direction (antiparallel DNA strands)DNA pol III catalyzes chain elongation only in the 5’3’ direction (antiparallel DNA strands) Leading strand - synthesized by polymerization in the same direction as fork movementLeading strand - synthesized by polymerization in the same direction as fork movement Lagging strand - synthesized by polymerization in the opposite direction of fork movementLagging strand - synthesized by polymerization in the opposite direction of fork movement Two core complexes of DNA pol III, one for leading, one for lagging strand

Replisome DNA synthesis Two core complexes of DNA pol III one for leading, one for lagging strand Replisome = primosome+ DNA Pol

Leading strand is synthesized as one continuous polynucleotide (beginning at origin and ending at the termination site)Leading strand is synthesized as one continuous polynucleotide (beginning at origin and ending at the termination site) Lagging strand is synthesized discontinuously in short pieces (Okazaki fragments)Lagging strand is synthesized discontinuously in short pieces (Okazaki fragments) Pieces of the lagging strand are then joined by a separate reaction By Pol I and DNA ligaseBy Pol I and DNA ligase fork 5’ 3’ fork 5’ 3’ Lagging-Strand Synthesisis Discontinuous Overall the process is semidiscontinuous

Demonstration of discontinuous DNA synthesisDemonstration of discontinuous DNA synthesis (In lagging strand) Pulse label E. coli by short time-period With 3 H-dTTP

(From previous slide)

RNA Primer Begins Each Okazaki Fragment Primosome is a complex containing primase enzyme which synthesizes short pieces of RNA at the replication fork (complementary to the lagging- strand template)Primosome is a complex containing primase enzyme which synthesizes short pieces of RNA at the replication fork (complementary to the lagging- strand template) DNA pol III uses the RNA primer to start the lagging- strand DNA synthesisDNA pol III uses the RNA primer to start the lagging- strand DNA synthesis Replisome - includes primosome, DNA pol III DNA pol can’t begin de novo (needs existing 3’OH) (needs existing 3’OH) DNA dependent RNA pol Now need to join Okazaki fragments

Joining of Okazaki fragments by DNA pol I and DNA ligase Remove RNA primer Synthesis replacement DNA Seal 2 DNA fragments Three steps:

Single polypeptide has both activities

Removal of RNA primer essential DNA ligase only uses dsDNA

DNA ligase mechanism

Klenow (large) fragment of DNA pol I, lacks 5’-3’ exonuclease activityKlenow (large) fragment of DNA pol I, lacks 5’-3’ exonuclease activity Pol I can be cleaved into Large frag: pol and proofreading (3’-5’ exo) Small frag: 5’-3’ exonuclease First enzyme found to syn DNA Simple enzyme used to syn DNA in the test tube

Replisome DNA synthesis Helicase in primase unwinds DNA DNA syn coupled to unwinding parental DNA But there is no large stretch of ssDNA SSB

SSB keeps DNA free of 2nd structure (good template) (good template) Unwinding assisted by topoisomerases Relieve supercoiling Not part of replisome topoII: gyrase Lagging strand encounters okazaki frag Releases lagging strand

Replisome assembles at origin site (oriC)Replisome assembles at origin site (oriC) Initial assembly depends on local unwinding of the DNA caused by binding certain proteinsInitial assembly depends on local unwinding of the DNA caused by binding certain proteins DnaA is one initiation proteinDnaA is one initiation protein Terminator utilization substance (Tus) binds to the ter siteTerminator utilization substance (Tus) binds to the ter site Tus inhibits helicase activity and thus prevents replication forks continuing through this regionTus inhibits helicase activity and thus prevents replication forks continuing through this region Initiation and Termination of DNA Replication E. coli

Eukaryotic cell cycle: regulation of replication S phase: DNA replication M phase: Mitotic cell division Only replicate once: One Drosophila chrom 600 replication forks Origins only used once! During S phase M-G1: ORC assembly at each Ori formation of pre-initiation complex S phase: replisomes “fire” in response to S-phase protein kinase high levels of kinase prevents re-loading of complexes 1N 2N After G2 the kinase is cleared and ORC assemble on new chromosome

DNA repair mechanisms The only cellular macromolecule that can be repaired Cost to organism (mutated or damaged DNA) far outweigh the energy of repair Single-celled organism- one mutation can kill DNA damage: base modifications, nucleotide deletions x-link DNA strands Repair protects individual cells and subsequent generations “oxygen catastrophe” introduced oxidative stress to nucleic acid system

DNA repair mechanisms Specific repair enzymes scan DNA to detect any alterationsSpecific repair enzymes scan DNA to detect any alterations Lesions may be fixed by direct repair, which does not require breaking the phosphodiester backbone of DNA or by more complicated reactionsLesions may be fixed by direct repair, which does not require breaking the phosphodiester backbone of DNA or by more complicated reactions The only cellular macromolecule that can be repaired Single-celled organism- one mutation can kill DNA damage: base modifications, nucleotide deletions x-link DNA strands Repair protects individual cells and subsequent generations Cost to organism (mutated or damaged DNA) far outweigh the energy of repair

Double-helical DNA is very sensitive to damage by UV lightDouble-helical DNA is very sensitive to damage by UV light Dimerization of adjacent pyrimidines in a DNA strand is common (e.g. thymines)Dimerization of adjacent pyrimidines in a DNA strand is common (e.g. thymines) Replication cannot proceed in the presence of pyrimidine dimers (template strand is distorted)Replication cannot proceed in the presence of pyrimidine dimers (template strand is distorted) Thymine dimers are repaired in all organismsThymine dimers are repaired in all organisms Repair after Photodimerization: An Example of Direct Repair

Repair of thymine dimers by DNA photolyaseRepair of thymine dimers by DNA photolyase

photoreactivation Direct repair: DNA backbone not cleaved

Excision Repair DNA can be damaged by alkylation, methylation, deamination, loss of heterocyclic bases (depurination or depyrimidization)DNA can be damaged by alkylation, methylation, deamination, loss of heterocyclic bases (depurination or depyrimidization) General excision-repair pathway can repair many of these defectsGeneral excision-repair pathway can repair many of these defects Overall pathway is similar in all organismsOverall pathway is similar in all organisms UvrABC endonuclease nucleotide gap

Hydrolytic deamination of cytosine to uracilHydrolytic deamination of cytosine to uracil Uracil in place of cytosine causes incorporation of an incorrect base during replicationUracil in place of cytosine causes incorporation of an incorrect base during replication DNA glycosylases hydrolyze base-sugar N-glycosidic bonds Deaminated bases are then removed and replaced Most common type of DNA damage Explains why uracil not part of DNA so this reaction will be recognized as an error

Repair of damage from deamination of cytosine Flips base out of helix and hydrolyzes Apurinic and apyrimidimic site AP endonuclease Base excision repair Uracil N-glycosylase

DNA repair by recombination 1. Repair 2. Exchange info Similar proteins utilized in DNA repair

Homologous Recombination Recombination - exchange or transfer of pieces of DNA from one chromosome to another or within a chromosomeRecombination - exchange or transfer of pieces of DNA from one chromosome to another or within a chromosome Homologous recombination - occurs between pieces of DNA that have closely related sequencesHomologous recombination - occurs between pieces of DNA that have closely related sequences Nonhomologous recombination occurs between unrelated sequences (e.g. Transposons )Nonhomologous recombination occurs between unrelated sequences (e.g. Transposons ) 1. Repair 2. Exchange info

Strand invasion Branch migration RecBCD endonuclease RecA RuvA and B RuvC E. coli proteins: Resolution The Holliday Model of General Recombination Cleavage and ssDNA formation

Recombination starts with generation of single- stranded DNA with a free 3’ end RecBCD endonuclease binds to DNA, cleaves one strand, then unwinds DNA in an ATP-dependent reaction Strand exchange begins when single-stranded DNA invades a neighboring double helix Rec A is a strand exchange protein Promote triple-strand interaction Recognize sequence homology Catalyzes strand exchange RecA catalyzes strand exchange E. coli

Action of Ruv proteins at Holliday junctionsAction of Ruv proteins at Holliday junctions

Fig Branch migration and resolution

Fig Model of RuvA and RuvB bound to Holliday junction