Chapter 10: The Mutability and Repair of DNA PDF

Chapter 10. The mutability and repair of DNA Outline 1. Replication errors and their repair 2. DNA damage 3. Repair and tolerance of...

Chapter 10. The mutability and repair of DNA Outline 1. Replication errors and their repair 2. DNA damage 3. Repair and tolerance of DNA damage © 2014 Pearson Education, Inc.  Low mutation rate critical for the perpetuation of the genetic material from generation to generation Mutations at protein coding sequence or DNA regions that control mRNA expression  change phenotypes of a cell Changes in DNA sequence or genetic variation Also important for driving evolution  New species including human  Biodiversity depends on a happy balance between mutation and repair Important sources of mutations 1) Inaccuracy in DNA replication 2) Chemical damage 3) Insertion of DNA elements(Transposons) © 2014 Pearson Education, Inc.  Errors in DNA replication and damage to DNA 1) Permanent changes to the DNA(mutations) 2) Prevent DNA replication or transcription In this chapter 1) How the alteration to the genetic material is detected 2) How this alteration is properly repaired and tolerated 3) How does the cell distinguish the parental strand from the daughter strand 4) How does the cell restore the proper DNA sequence when original sequence can no longer be read? © 2014 Pearson Education, Inc.  Replication errors and their repair The Nature of mutations The simplest mutations 1) Transitions : - pyrimidine pyrimidine, - purinepurine - T  C, A  G 2) Transconversions : - pyrimidine  purine, - purinepyrimidine - T  G or A, A  C or T 3) Insertions or deletions of a single nucleotide  Point mutation © 2014 Pearson Education, Inc.  Extensive insertions or deletions and gross rearrangements of chromosome structure  cause more drastic changes in DNA Spontaneous mutation rate - 10-6 ~ 10-11 per round of DNA replication - “Hot spot” : mutations arise at high frequency ex) DNA microsatellites : repeats of di-, tri-, or tetranucleotide sequences  CA repeats - many scattered sites in the genomes of eukaryotes - difficult to copy accurately  increase or decrease the number of copies  highly polymorphic  can be used as a marker for inherited mutations © 2014 Pearson Education, Inc. Figure 10-1 Some replication errors escape proofreading DNA replication machinery has a proofreading function - 3’  5’ exonuclease activity - is not foolproof - Some misincorporated nts escape detection  mismatch  permanent change in the DNA sequence © 2014 Pearson Education, Inc. Figure 10-2 Mismatch repair removes errors that escape proofreading Mismatch repair system - Increases the accuracy of DNA synthesis Two challenges 1) Must rapidly scan the genome for mismatches 2) Must correct the mismatch accurately (the newly synthesized strand) In E. coli MutS dimer - detects mismatches (DNA is more readily distorted)  induces a conformational change in MutS itself - recruits MutL, a second component of the repair system - MutL activates MutH, an enzyme that causes an incision or nick © 2014 Pearson Education, Inc. Figure 10-3 © 2014 Pearson Education, Inc.  Which of the two mismatched nucleotides should be replaced? Dam methylase adds methyl group on A residues - 5’-GATC-3’ (can be found about once every 256bp) - Methylates on both strands of parental strands (fully methylated) - After DNA synthesis and until Dam methylates new DNAs  only one parental strand is methylated (hemimethylated) MutH binds at hemimethylated sites  MutL and MutS activate endonuclease activity of MutH  MutH nicks the unmethylated strand © 2014 Pearson Education, Inc. Figure 10-5 Dam methylation at replication fork © 2014 Pearson Education, Inc.  Different exonucleases are used to remove single-stranded DNA between the nick and the mismatch If DNA is cleaved on 5’ side of the mismatch  Exonuclease VII or RecJ  Degrades DNA in a 5’ 3’ direction If the nick is on 3’ side  Exonuclease I  Degrades DNA in a 3’ 5’ direction © 2014 Pearson Education, Inc. Figure 10-6 Directionality in mismatch repair: Exonuclease removal of mismatched DNA © 2014 Pearson Education, Inc.  In eukaryotic cells - MSH protein (homolog of MutS) - MLH and PMS (homolog of MutL) - Multiple MutS-like proteins 1) specific for simple mismatches 2) small insertions or deletions - Mutation of MutS and MutL in higher organisms  causes colon cancer Eukaryotic cells don’t have MutH and Dam methylase  Okazaki fragments are separated by nicks  This strand can be repaired MSH(MutS) interacts with the sliding clamp  Recruits mismatch repair proteins to the lagging and leading strand © 2014 Pearson Education, Inc.  DNA damage DNA undergoes damage spontaneously from hydrolysis and deamination Mutations - from errors in replication - from damage to DNA DNA damage can be occurred by 1) Environmental factors (radiation) 2) Mutagens which are chemical agents that increase mutation frequency © 2014 Pearson Education, Inc. Box 10-2-1 The Ames Test - identify the potential mutagens © 2014 Pearson Education, Inc.  Deamination of the base cytosine - The most frequent and important kind of hydrolytic damage - Spontaneously occurs under normal condition  Generates the unnatural base uracil (in DNA) NH3 C U G A Deamination of Adenine and Guanine Adenine  hypoxanthine (hydrogen bonds to cytosine) Guanine  xanthine (pairs with cytosine with only two hydrogen bonds) © 2014 Pearson Education, Inc.  In contrast to the replication errors -these hydrolytic reactions result in unnatural alterations of the DNA  Can be recognized by DNA repair enzyme Why DNA has thymine instead of uracil? If DNA has uracil, deamination of cytocine generates a natural base  Can not be repaired by the repair systems 5-methylcytosine (in higher euk.) - By methyltransferase - Involved in transcriptional silencing - Deamination generates thymine - CT transition © 2014 Pearson Education, Inc.  DNA is damaged by alkylation, oxidation, and radiation Alkylation - Methyl or ethyl groups are transferred to reactive sites on the bases - Nitrosamine and nitrosoguanidine - Generates methylguanine  mispairs with thymine Oxidation - By reactive oxygen species (O-2, H2O2, OH ) - Oxidation of guanine generates oxoguanine  base-pairs with adenine and cytosine © 2014 Pearson Education, Inc.  Ultraviolet (UV) - Radiation(260nm) is strongly absorbed by the bases  the photochemical fusion of two pyrimidines  thymine dimer (cyclobutane ring)  thymine-cytosine fusion  cause termination of DNA replication γ-radiation and X-rays - Cause double-strand breaks in the DNA - Can be lethal to a cell Bleomycin (anticancer drug) - Causes breaks in DNA and kills rapidly growing cells - Clastogenic agents (Greek Klastos means “broken”) © 2014 Pearson Education, Inc.  Mutations are also caused by base analogs & intercalating agents - Cause errors in DNA replication Base analogs - Similar structure to proper bases - Incorporated into DNA during replication - base-pair inaccurately - 5-bromouracil – thymine analog © 2014 Pearson Education, Inc.  Intercalating agents - Contain several polycyclic ring - Cause additions or deletions of a base pair Insertion - Slipping between bases in the template strand  DNA polymerase inserts an extra nucleotide Deletion - The distortion to the template by the presence of an inserted molecule  DNA polymerase might skip a nucleotide © 2014 Pearson Education, Inc.  Repair and Tolerance of DNA damage Two consequences of DNA damage 1) Thymine dimers, nicks and breaks in the DNA - block DNA replication and transcription 2) Deamination of cytosine…… - C:G  T:A transition mutation  Cells must repair DNA damage before it blocks replication or causes a mutation 1) Excision repair - removes the damaged nucleotide from the DNA 2) Recombinational repair (double-strand break repair) - both strands are damaged 3)Translesion Polymerase - synthesize DNA across the site of the damage © 2014 Pearson Education, Inc. Table 10-1 © 2014 Pearson Education, Inc. Figure 10-11 Direct reversal of DNA damage 1) Photoreactivation(simple reversal of damage) 2) Removal of the methyl group from methylguanine 1) Photoreactivation - Photolyase captures energy from light and uses it to break the covalent bonds linking pyrimidines © 2014 Pearson Education, Inc. Figure 10-12 2) Removal of the methyl group from methylguanine - a methyltransferase removes the methylgroup from G - transfer it to one of its own cysteine residues - the methyltransferase accepted a methyl group can not be used again © 2014 Pearson Education, Inc.  Base excision repair enzymes remove damaged bases by a base-flipping mechanism - Remove and replace the altered bases - Base excision repair and nucleotide excision repair 1) Base excision repair - Glycosylase hydrolyzes the glycosidic bond  abasic sugar  removed from DNA by endonuclease  restored by DNA pol and DNA ligase © 2014 Pearson Education, Inc.  Glycosylase - 11 different DNA glycosylases in human - different specificities 1) one recognizes uracil(generated by deamination of cytosine) 2) another removes oxoG(oxidation of guanine) If a damaged base is not removed by base excision,  oxoG:A pair - a dedicated glycosylase(a fail-safe system) removes the A - the A is the undamaged but incorrect base © 2014 Pearson Education, Inc.  Nucleotide excision repair enzymes cleave damaged DNA on either side of the lesion - Recognizes distortions to the shape of the double helix : a thymine dimer or a bulky chemical adduct on a base - Removes a short single-strand segment that includes the lesion In E. coli - 4 proteins : UvrA, UvrB, UvrC, and UvrD 1) UvrA and UvrB  scan the DNA 2) UvrC  creates two incisions  one located 4~5 nts 3’ and the other 8 nts 5’ to the lesion  a 12~13-residue-long DNA strand 3) UvrD (DNA helicase)  removes the lesion-containing strand  generates a small gap © 2014 Pearson Education, Inc. Figure 10-16 © 2014 Pearson Education, Inc.  Nucleotide excision repair in eukaryotes - Very similar to those of E. coli - More than 25 proteins are involved 1) XPC  detects distortions to the helix 2) XPA and XPD  form a bubble by helicase activity 3) RPA  single stand binding protein 4) ERCC1-XPF  cuts 5’ to the lesion 24~32 nucleotides 5) XPG  cuts 3’ to the lesion © 2014 Pearson Education, Inc. Figure 10-17 Transcription-coupled repair - RNA polymerase also scans DNA damage - If DNA is damaged  RNA polymerase stalls and transcription stops - RNA polymerase recruits nucleotide excision repair proteins TFIIH - A key protein of transcription-coupled repair - Unwinds the DNA template - Includes XPA and XPD(helicase activity) Two separate functions 1) During repair - helicase melt the DNA around a lesion 2) Gene transcription - open the DNA template © 2014 Pearson Education, Inc. Figure 10-17 © 2014 Pearson Education, Inc.  Recombination repairs DNA breaks by retrieving sequence information from undamaged DNA - Excision repair uses the undamaged DNA as a template How do cells repair double-strand breaks in DNA?  Double-strand break(DSB) repair (Chapter 11) DSBs in DNA are also repaired by direct joining of broken ends - DSB is the most cytotoxic of all DNA damage - Blocks DNA replication and causes chromosome loss  cell death Non-homologous end joining (NHEJ) - Protects and processes the broken ends and then joins them together - Sequence information is lost from the ends - Mutagenic because the original sequence is not fully restored © 2014 Pearson Education, Inc.  Mechanism of Non-homologous end joining (NHEJ) - Seven proteins are involved in NHEJ - Ku70, Ku80, DNA-PKcs, Artemis, XRCC4, Cernunnos-XLF and DNA ligase IV 1) Ku70 and Ku80 : heterodimer - Binds to the DNA ends - Recruits DNA-PKcs, a protein kinase 2) DNA-PKcs - Form a complex with Artemis 3) Artemis - 5’3’ exonuclease and endonuclease - Activated by phosphorylation by DNA-PKcs - Process the broken DNA and prepare them for ligation 4) Ligase IV + XRCC4 + XLF - Performs ligation © 2014 Pearson Education, Inc. Figure 10-18 © 2014 Pearson Education, Inc.  Translesion DNA synthesis enables replication to proceed across DNA damage Once DNA pol encounters a lesion that has not been repaired  Replication machinery can bypass these sites  Tolerate the DNA damage Translesion synthesis - One mechanism of DNA damage tolerance - Highly error-prone - Introduces mutations - The lesion remains in the genome  can be corrected by DNA repair system © 2014 Pearson Education, Inc. © 2014 Pearson Education, Inc.  Translesion synthesis - Catalyzed by a specialized class of DNA polymerase - Synthesizes DNA directly across the site of the damage In E. coli - DNA pol IV(DinB) or DNA pol V(a complex of UmuC and UmuD’)  Y family of DNA polymerase - Template-dependent  don’t read sequence information from the template - Incorporate nucleotides in a manner that is independent of base pairing But, some translesion pols incorporate specific nucleotides Ex) human DNA pol η  inserts two A residues opposite a thymine dimer © 2014 Pearson Education, Inc. Figure 10-20 © 2014 Pearson Education, Inc.  Translesion synthesis - Highly mutagenic  Translesion DNA polymerase must be tightly regulated In E. coli -Translesion DNA polymerases are not expressed under normal condition - Induced by DNA damage “SOS response” - DNA damage causes degradation of transcriptional repressor, LexA that inhibits expression of DinB, UmuC, and UmuD © 2014 Pearson Education, Inc.  How a translesion pol accesses to the stalled replication machinery at the site of DNA damage? In higher eukaryotes -Triggered by chemical modification of the sliding clamp  Ubiquitination Once ubiquitinated, The sliding clamp recruits a translesion pol containing domains that recognize and bind to ubiquitin Displaces the replicative polymerase from 3’ end of the growing strand “Polymerase switching” “Gap filling mechanism” © 2014 Pearson Education, Inc. Figure 10-23 © 2014 Pearson Education, Inc. Table 10-1 © 2014 Pearson Education, Inc.