Chapters 16, 15 & 17 Genetic Recombination & DNA Repair Types of mutations discussed -thus far Nonsense mutation: A mutation in which a nucleotide change in DNA results when a “sense codon" that corresponds to one of the twenty amino acids specified by the genetic code is changed to a STOP-codon. Missense mutation A point mutation in which a single nucleotide change results in a codon that codes for a different amino acid. It is a type of nonsynonymous substitution. • synonymous mutation – A change in DNA sequence in a coding region that does not alter the corresponding amino acid that is encoded. • non synonymous mutation – A change in DNA sequence in a coding region that does alter the amino acid that is encoded. Tautomeric shifts invariably give rise to transitions. Repair Systems Correct Damage to DNA • mismatch repair (MMR) – A type of repair that corrects mispaired bases, typically immediately following replication. – The process preferentially corrects the sequence of the daughter strand by distinguishing the daughter strand and parental strand, sometimes on the basis of their states of methylation. MutSL binds mismatches on unmethylated DNA strands DNA damage products formed after attack by oxygen radicals. Repair Systems Correct Damage to DNA • mismatch repair (MMR) – A type of repair that corrects mispaired bases, typically immediately following replication. – The process preferentially corrects the sequence of the daughter strand by distinguishing the daughter strand and parental strand, sometimes on the basis of their states of methylation. Base Excision Repair Systems Require Glycosylases • Base excision repair is triggered by directly removing a damaged base from DNA. • Base removal triggers the removal and replacement of a stretch of polynucleotides. • The nature of the base removal reaction determines which of two pathways for excision repair is activated. • The polδ/ε pathway replaces a long polynucleotide stretch; the polβ pathway replaces a short stretch (sometime “single”bp). Repair Systems Correct Damage to DNA • mismatch repair (MMR) – A type of repair that corrects mispaired bases, typically immediately following replication. – The process preferentially corrects the sequence of the daughter strand by distinguishing the daughter strand and parental strand, sometimes on the basis of their states of methylation. Repair Systems Correct Damage to DNA • mismatch repair (MMR) – A type of repair that corrects mispaired bases, typically immediately following replication. – The process preferentially corrects the sequence of the daughter strand by distinguishing the daughter strand and parental strand, sometimes on the basis of their states of methylation. • Photoreactivation – A repair mechanism that uses a white light-dependent enzyme to split cyclobutane pyrimidine dimers formed by ultraviolet light. • excision repair – A type of repair system in which one strand of DNA is directly excised and then replaced by re-synthesis using the complementary strand as template. Excision repair directly replaces damaged DNA and then resynthesizes a replacement stretch for the damaged strand. Nucleotide excision repair occurs via two major pathways: global genome repair, in which XPC recognizes damage anywhere in the genome, and transcription-coupled repair, in which the transcribed strand of active genes is preferentially repaired and the damage is recognized by an elongating RNA17 polymerase. 18 Repair Systems Correct Damage to DNA • Recombination repair (RR) – A type of repair that corrects mispaired bases, preferentially using other DNA strands as template for repair. Recombination-Repair Systems • The single strand of another duplex is used to replace the gap (single-strand exchange). • The damaged sequence is then removed and re- synthesized. Recombination-repair uses two duplexes RecA creates a recombination intermediate Role of RecA and other associated proteins in different recombinational events within the E. coli bacterial system. RecBCD aggregates and binds to a chi sequences (5'GCTGGTGG3'; which occurs ~every 70 kbp: [or ~65,536 bp] within the E. coli chromosome) and creates a "free" single stranded DNA end that can go and "seek out" similar DNA sequences with which to hybridize. Mark S. Dillingham, and Stephen C. Kowalczykowski Microbiol. Mol. Biol. Rev. 2008; doi:10.1128/MMBR.00020-08 Homologous Recombination involves a process in which the single strands in the region of the crossover exchange their partners. Figure 1.32 shows that this creates a stretch of hybrid DNA in which the single strand of one duplex is paired with its complement from the other duplex. Simple… 2 Stranded Break & ligation model -involves a process in which the single strands in the region of the crossover exchange their partners. Simple… 2 Stranded Break & ligation model -involves a process in which the single strands in the region of the crossover exchange their partners. Simple… 2 Stranded Break & ligation model -involves a process in which the single strands in the region of the crossover exchange their partners. Simple… 2 Stranded Break & ligation model -involves a process in which the single strands in the region of the crossover exchange their partners. Simple… 2 Stranded Break & ligation model -involves a process in which the single strands in the region of the crossover exchange their partners. Holliday (“double nick”) Meselson-Radding (“single nick”) Holliday (“double nick”) Meselson-Radding (“single nick”) Branch migration, the movement of the crossover point between DNA complexes that is catalyzed by the RuvAB complex. Then (in E. coli)……..RuvC acts as the resolvase and cleaves two of the DNA strands -demonstrating a preference for 5’a/tTT - GC3’ Figure 19-11. (a) The Holliday structure shown in an extended form. (b) The rotation of the structure shown in part a can yield the form depicted in part c. Resolution of the structure shown in part c can proceed in two ways, depending on the points of enzymatic cleavage, yielding the structures shown in part d. The dotted lines show which segments will rejoin to form recombinant strands for each particular cleavage scheme. The strands are shown linearly in part e and can be repaired to the forms shown in part f. (From H. Potter and D. Dressler, Cold Spring Harbor Symposium on Quantitative Biology 43, 1970, 970. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.) (a) A duplex is cut on one chain. (b) DNA polymerase displaces one chain. (c) The resulting single chain displaces its counterpart in the homolog. (d) This displaced chain is enzymatically digested. (e) Ligation completes the formation of a Holliday junction. (f) Resolution of the junction occurs as in the Holliday model. The Meselson-Radding “single stranded break” or “heteroduplex” model. In both the original Holliday and Meselson-Radding models for genetic recombination, the initiation events for recombination are single-strand nicks that result in the generation of heteroduplex DNA. However, the finding in yeast was that transformation is stimulated 1,000- fold when a double-strand break is introduced into a circular donor plasmid .... giving rise to an additional model, the double-strand-break DSB model, originally formulated by Jack Szostak, Terry Orr-Weaver, Rodney Rothstein and Franklin Stahl. Break Induced Replication (BIR) initiates translocations (is this example peri- or para- centric) Fig. 1. Current DSB-initiated model of homologous recombination (modified from [9]). Double-strand break-repair model of homologous recombina@on. • The RecA homolog Rad51 forms a nucleoprotein filament on the single-stranded regions, assisted by Rad52 and Rad55/57. • Rad54 and Rdh54/Rad54B are involved in homology search and strand invasion. • The yeast RAD mutations were identified by radiation-sensitive phenotypes and are in genes that code for repair systems. • The RecA homolog Rad51 forms a nucleoprotein filament on the single-stranded regions, assisted by Rad52 and Rad55/57. • Rad54 and Rdh54/Rad54B are involved in homology search and strand invasion. • The yeast RAD mutations were identified by radiation-sensitive phenotypes and are in genes that code for repair systems. http://mcb.asm.org/cgi/reprint/27/5/1868 Spo11 and other endonucleases generate double-strand breaks. • The RAD52 group of genes (which includes Rad50) is required for recombination repair, and is inter\gral to the formation of synaptonemal complexes in yeast • The MRX (yeast) or MRN (mammals) complex is required to form a single- stranded region at each DNA end. The MRN complex, required for 5’ end resec@on, also serves as a DNA bridge to prevent broken ends from separa@ng. Don’t forget that DNA Repair in Eukaryotes Occurs in the Context of Chromatin • Different patterns of histone modifications may distinguish stages of repair or different pathways of repair. • Remodelers and chaperones are required to reset chromatin structure after completion of repair. Meiotic vs. Mitotic recombination? Homologous recombination is a reaction between two duplexes of DNA. Its critical feature is that the enzymes responsible can use any pair of homologous sequences as substrates (although some types of sequences may be favored over others). The frequency of recombination is not constant throughout the genome, but is influenced by both global and local effects. Homologous recombination is a reaction between two duplexes of DNA. Its critical feature is that the enzymes responsible can use any pair of homologous sequences as substrates (although some types of sequences