DNA Repair and Recombination Lecture 8a PDF

Lecture 8a DNA repair and recombination Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 25: p. p. 930-943 DNA METABOLISM (2) Key topics: – DNA repair – DNA recombination DNA REPAIR AND MUTATIONS Nucleotides and nucleic acid undergo alterations in th...

Lecture 8a DNA repair and recombination Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 25: p. p. 930-943 DNA METABOLISM (2) Key topics: – DNA repair – DNA recombination DNA REPAIR AND MUTATIONS Nucleotides and nucleic acid undergo alterations in their structure, leading to mutations in the DNA sequence, that are linked to cancer Chemical reactions and some physical processes constantly damage genomic DNA - The majority are corrected using the undamaged strand as a template. - Some base changes escape repair and an incorrect base serves as a template in replication. - The daughter DNA carries a changed sequence in both strands Accumulation of mutations in eukaryotic cells is strongly correlated with cancer; most mutagens are also carcinogens Thousands of lesions/day but only 1/1000 becomes a mutation, thanks to DNA repair Human genome contains genes for > 130 repair proteins VOCABULARY OF DNA LESIONS Lesion = DNA damage – If unrepaired, a lesion becomes a mutation – Mutations can be substitutions (point mutations), deletions, additions. A silent mutation―has almost no effect on gene function THE AMES TEST INDICATES THE MUTAGENIC POTENTIAL OF A COMPOUND Developed by Bruce Ames Uses Salmonella strain with a mutation that makes bacterium unable to synthesize His (a mutation that inactivates an enzyme of the histidine biosynthetic pathway) Add compound to plate of Salmonella, see if it grows in His-free medium – Colonies (+ test) indicates the compound mutated the Salmonella, restored ability to synthesize His THE AMES TEST FOR CARCINOGENS Ames test for carcinogens, based on their mutagenicity. A strain of Salmonella typhimurium having a mutation that inactivates an enzyme of the histidine biosynthetic pathway is plated on a histidine-free medium. Few cells grow. (a) The few small colonies of S. typhimurium that do grow on a histidine-free medium carry spontaneous back-mutations that permit the histidine biosynthetic pathway to operate. Three identical nutrient plates (b), (c), and (d) have been inoculated with an equal number of cells. Each plate then receives a disk of filter paper containing progressively lower concentrations of a mutagen. The mutagen greatly increases the rate of backmutation and hence the number of colonies. The clear areas around the filter paper indicate where the concentration of mutagen is so high that it is lethal to the cells. As the mutagen diffuses away from the filter paper, it is diluted to sub-lethal concentrations that promote back-mutation. Mutagens can be compared on the basis of their effect on mutation rate. Because many compounds undergo a variety of chemical transformations after entering cells, compounds are sometimes tested for mutagenicity after first incubating them with a liver extract. Some substances have been found to be mutagenic only after this treatment. HOW CHEMICAL MODIFICATIONS OF NUCLEOTIDES PRODUCE MUTATIONS If not repaired, this leads to a G≡C to A=T mutation after replication The methylation product O6-methylguanine pairs with thymine rather than cytosine. HOW CHEMICAL MODIFICATIONS OF NUCLEOTIDES PRODUCE MUTATIONS (A) Deamination of cytosine, if uncorrected, results in the substitution of one base for another when the DNA is replicated (deamination of cytosine produces uracil). Uracil differs from cytosine in its basepairing properties and preferentially base-pairs with adenine. The DNA replication machinery therefore adds an adenine when it encounters a uracil on the template strand. (B) Depurination can lead to the loss of a nucleotide pair. When the replication machinery encounters a missing purine on the template strand, it may skip to the next complete nucleotide as illustrated here, thus producing a nucleotide deletion in the newly synthesized strand. Many other types of DNA damage (see Figure 5–37), if left uncorrected, also produce mutations when the DNA is replicated. TYPES OF DNA DAMAGE Mismatches arise from occasional incorporation of incorrect nucleotides Abnormal bases arise from spontaneous deamination, chemical alkylation or exposure to free radicals Pyrimidine dimers form when DNA is exposed to UV light Backbone lesions occur from exposure to ionizing radiation, free radicals CELLS HAVE MULTIPLE DNA REPAIR SYSTEMS MISMATCH REPAIR RELIES ON METHYLATION (1) After replication, the newly synthesized strand is unmethylated for a short period of time after synthesis Any replication errors must reside in the unmethylated strand Methyl-directed mismatch repair system cleaves the unmethylated strand in the initial part of the repair process… MISMATCH REPAIR RELIES ON METHYLATION (2) How do repair enzymes “know” which strand is the correct one? Methylation of DNA strands can serve to distinguish parent (template) strands from newly synthesized strands in E. coli DNA. This function is critical to mismatch repair. In E. coli, the parent strand is methylated whereas the new strand is not: This DNA is called hemi-methylated DNA. Mismatch repair occurs during the time the DNA is hemi-methylated. After that, the daughter strand is then methylated. Methylation is accomplished by Dam methylase: inserts CH3 at N6 of adenines in GATC sequence.This sequence is a palindrome, present in opposite orientations on the two strands.. EARLY STEPS OF METHYL-DIRECTED MISMATCH REPAIR IN E. COLI Mut S scans the DNA for mismatches MutL forms a complex with MutS MutL and MutS complex binds to the mismatch in the DNA MutH protein binds at hemi-methylated GATC nearby DNA on both side of the mismatch is threaded through the complex, creating a DNA loop, until methylated MutH-GATC is encountered – Mismatch could be 1000 bp away from GATC à high energy cost! METHYL-DIRECTED MISMATCH REPAIR IN E. COLI: EARLY STEPS The MutL protein forms a complex with MutS at the mismatch. DNA is threaded through this complex such that the complex slides along the DNA until it encounters a MutH protein bound at a hemi-methylated GATC sequence. Interactions between MutL-MutS complex and MutH activates MutH endonuclease activity à MutH cleaves the unmethylated strand on the 5′ side of the G in this sequence. Recognition of the sequence (5′)GATC is a specialized function of MutH Recognition of the mismatch is a specialized function of MutS proteins. COMPLETION OF METHYL-DIRECTED MISMATCH REPAIR IN E. COLI MutH cleaves non-methylated DNA strand on the 5’side of the G Futher steps of the pathway depend on where the mismatch is located relative to the cleavage site. – Mismatch on the 5’ side: DNA unwinds and is degraded 3’à5’ Helicase II (UvrD helicase) and exonucleases work to degrade the non-methylated DNA toward the mismatch The removed sequence is replaced using DNA Pol III and DNA ligase – Mismatch on the 3’side: Same mechanism, except other enzymes are used. COMPLETION OF METHYL-DIRECTED MISMATCH REPAIR IN E. COLI The combined action of DNA helicase II, SSB, and one of four different exonucleases removes a segment of the new strand (unmethylated DNA strand) between the MutH cleavage site and a point just beyond the mismatch. The particular exonuclease that is used depends on the location of the cleavage site relative to the mismatch, as shown by the alternative pathways here. The resulting gap is filled in (dashed line) by DNA polymerase III, and the nick is sealed by DNA ligase (not shown here). NICK-DIRECTED MISMATCH REPAIR IN EUKARYOTES No MutH in eukaryotes: Therefore, it is the interaction between MutS:MutL complex and replication prcessivity factor (bclamp) that activates the latent MutL endonuclease to nick the error-containing daughter strand (on both sides of the error). (PNAS:, 112, 10914-10919) (A)The two proteins shown are present in both bacteria and eukaryotic cells: MutS binds specifically to a mismatched base pair, while MutL scans the nearby DNA for a nick. Once MutL finds a nick, it triggers the degradation of the nicked strand all the way back through the mismatch. Because nicks are largely confined to newly replicated strands in eukaryotes, replication errors are selectively removed. In bacteria, an additional protein in the complex (MutH) nicks unmethylated (and therefore newly replicated) GATC sequences, thereby beginning the process illustrated here. In eukaryotes, MutL contains a DNA nicking activity that aids in the removal of the damaged strand. (B)The structure of the MutS protein bound to a DNA mismatch. This protein is a dimer, which grips the DNA double helix as shown, kinking the DNA at the mismatched base pair. It seems that the MutS protein scans the DNA for mismatches by testing for sites that can be readily kinked, which are those with an abnormal base pair. BASE EXCISION REPAIR USES SPECIFIC DNA GLYCOSYLASES DNA glycosylases (found in all kingdoms of life) recognize specific lesions à Cleave N-glycosidic bond between the sugar and the base, thus creating an apurinic/apyrimidinic (AP) site Uracil glycosylase removes uracil from DNA making an AP site Important because C spontaneously deaminates to U, and it needs to be repaired; That may be the main reason why U does not belong in DNA Deamination is 100´ faster in ssDNA Other glycosylases make AP sites – At 8-hydroxyG, hypoxanthine, 3-methyladenine, etc. REPAIR AT AP SITES IN BACTERIA The entire nucleotide is ultimately removed, not just the damaged base – In fact, sometimes the region around the AP site is removed AP endonucleases cut the DNA backbone around the AP site (makes a nick in the DNA) DNA Pol I synthesizes new DNA (nicktranslation activity) DNA ligase seals the nick BASE-EXCISION REPAIR (RECAP) 1 A DNA glycosylase recognizes a damaged base and cleaves between the base and deoxyribose in the backbone. 2 An AP endonuclease cleaves the phosphodiester backbone near the AP site. 3 DNA polymerase I initiates repair synthesis from the free 3′ hydroxyl at the nick, removing (with its 5′→3′ exonuclease activity) and replacing a portion of the damaged strand. 4 The nick remaining after DNA polymerase I has dissociated is sealed by DNA ligase.