8b-Biochemistry-Lecture8b.pdf

Lecture 8b DNA repair and recombination Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 25: p. p. 930-943 and 944-956 LARGE DISTORTIONS IN DNA ARE REPAIRED BY NUCLEOTIDE EXCISION Lesions include: – Cyclobutane pyrimidine dimers (CPDs) (formed from U...

Lecture 8b DNA repair and recombination Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 25: p. p. 930-943 and 944-956 LARGE DISTORTIONS IN DNA ARE REPAIRED BY NUCLEOTIDE EXCISION Lesions include: – Cyclobutane pyrimidine dimers (CPDs) (formed from UV light) – pyrimidine-pyrimidone 6,4-photoproducts (formed from UV light) UV irradiation mostly damages DNA by forming dimers at dipyrimidine sites, and ∼100,000 to 200,000 DNA lesions are generated per diploid cell in human skin by a moderate dose of UV (1 h of sunlight; equivalent to ∼30 mJ/cm2 UVB [280 to 320 nm])* – benzo[a]pyreneguanine (from cigarette smoke) à stable N2-dG adduct à mutations à cancer Repair Pathway involves removal of a DNA segment by Excinucleases DNA photoproducts formed by ultraviolet radiation. (a) Normal dTpT; (b) cis-syn cyclobutane pyrimidine dimer (CPD); (c) Oxetane intermediate; (d) A dT(6-4)T photoproduct with atom numbering; and (e) Dewar isomer. *PNAS, 2020 117 (23) 12806-12816 Int. J. Mol. Sci 15(11): 20321–20338. Cancer Res 18 2006 66 (24) 11938-11945 NUCLEOTIDE EXCISION REPAIR IN BACTERIA USES THE ABC EXCINUCLEASE Excinucleases cleave DNA backbone in two sites (dual incision) – ABC excinuclease contains UvrA, UvrB, and UvrC subunits: Hydrolyzes fifth bond on 3’-side of the lesion and eighth bond on 5’-side Helicase (UvrD) Removes 12–13 nucleotides DNA Pol I and DNA ligase replace the DNA and seal the gap NUCLEOTIDE EXCISION REPAIR IN EUKARYOTES Excinucleases, also used in eukaryotes, cleave DNA backbone in two sites – Hydrolyze sixth bond on 3’-side of the lesion and twenty-second bond on 5’-side Helicase Removes 27–29 nucleotides In humans, gap filled using DNA polymerase e and ligated by DNA ligase NUCLEOTIDE EXCISION REPAIR IN BACTERIA AND HUMANS 1 An excinuclease binds to DNA at the site of a bulky lesion and cleaves the damaged DNA strand on TWO sides of the lesion. 2 The DNA segment—of 13 nucleotides (13 mer) or 29 nucleotides (29 mer)—is removed with the aid of a helicase. 3 The gap is filled in by DNA polymerase I (bacteria) or Bacteria: Removal of 13 Nt Humans: Removal of 29 Nt DNA polymerase e (humans). 4 The remaining nick is sealed with DNA ligase. DIRECT REPAIR IN BACTERIA AND HUMANS Photolyases use light energy to repair pyrimidine dimers – Enzymes found in E. Coli but NOT in humans and placental mammals O6-methylguanine-DNA methyltransferase repairs methylated guanine to guanine – Enzyme found in humans AlkB demethylates 1-methyladenine and 3methylcytosine to adenine and cytosine – Enzyme found in humans DIRECT REPAIR OF PYRIMIDINE DIMERS WITH PHOTOLYASE IN E. COLI § Energy derived from absorbed light is used to reverse the photoreaction that caused the lesion. The two chromophores in E. coli photolyase, N5,N10methenyltetrahydrofolylpolygluta mate (MTHFpolyGlu) and FADH–, perform complementary functions. § MTHFpolyGlu functions as a photoantenna to absorb blue-light photons. The excitation energy passes to FADH–, and the excited flavin (*FADH–) donates an electron to the pyrimidine dimer, leading to the rearrangement as shown DIRECT REPAIR OF O6-METHYL GUANINE AND OTHER ALKYLATED BASES O6-methylguanine-DNA methyltransferase repairs methylated guanine to guanine The AlkB protein is an α-ketoglutarate-Fe2+– dependent dioxygenase. It catalyzes the oxidative demethylation of 1-methyladenine and 3-methylcytosine residues. WITHOUT DNA REPAIR, SPONTANEOUS DNA DAMAGE WOULD RAPIDLY CHANGE DNA SEQUENCES JC04 and JC05: DNA REPAIR DISEASES SOME INHERITED HUMAN SYNDROMES WITH DEFECTS IN DNA REPAIR WHAT HAPPENS WHEN THERE IS NO UNDAMAGED DNA TO USE AS A TEMPLATE FOR REPAIR? There is no template in situations of double strand breaks, cross-links, or damage in single strand DNA. Thus, – Unrepaired lesion can cause replication fork to stall Repair using 1) Error-Prone Translesion Synthesis (TLS) 2) Another chromosome as template (Recombination) DNA DAMAGE AND ITS EFFECT ON DNA REPLICATION Stalled Replication Fork Left: If the replication fork encounters an unrepaired lesion or strand break, the DNA Pol disengages and reinitiates downstream. The lesion remains in unreplicated, single strand gap that is left behind the replication fork. Replication generally halts and the fork may collapse. Double-strand break Right: In other cases, a replication fork may encounter a lesion that is actively undergoing repair such that a transient break is present in one of the template strands. When the replication fork encounters it, the single strand break becomes a double strand break. In each case (left and right) the damage to one strand cannot be repaired by mechanisms described earlier, because the complementary strand required to direct accurate repair is damaged or absent. Thus, two possible avenues for repair: - recombinational DNA repair or, - when lesions are unusually numerous, error-prone repair (or Trans Lesion Repair). - Trans Lesion Repair mechanism involves a novel DNA polymerase (DNA polymerase V, encoded by the umuC and umuD genes and activated by Rec A protein) that can replicate, albeit inaccurately, over many types of lesions. The repair mechanism is "error-prone" because mutations often result. ERROR-PRONE TRANSLESION DNA SYNTHESIS (TLS) AND DNA RECOMBINATION RECOMBINATION VS TRANSLESION REPAIR Infrequent lesions à Recombination Repair Frequent lesions à TLS Campbell and Farrell, 8th Ed. ERROR-PRONE TRANSLESION SYNTHESIS OF DNA IN BACTERIA TransLesion Synthesis (TLS) is part of the “SOS response” – Response when DNA damage is extensive SOS proteins include – UvrA and UvrB (UV radiation) – UmuC and UmuD (Umu = Unmutable, because lack of Umu genes abolishes the pathway) Cleaved UmuD’ and UmuC bind with RecA to create DNA Pol V (UmuD’2 –UmuC- RecA) – DNA Pol V possesses a more open active site (to accommodate the lesion) and can process past the damage but do not have proofreading activities. THE SOS RESPONSE IN E. COLI RecA homolog in mammals is Rad 51 Why have error-prone repair? It kills some cells, mutates DNA in others, but…some cells survive TLS POLYMERASES ABOUND IN MAMMALS Most recognize specific types of damage and have appropriate response Example: DNA Pol h (eta) in mammals assists when a T-T dimer halts a replication fork, and inserts two A residues opposite a T-T pyrimidine dimer, thus minimizing mutations Most TLS polymerases are limited to short regions of DNA, minimizing mutagenic potential IN EUKARYOTES, AS IN BACTERIA, TLS POLYMERASES USE DAMAGED TEMPLATES ubiquitination A replicative polymerase stalled at a site of DNA damage is recognized by the cell as needing rescue. Specialized enzymes covalently modify the sliding clamp (typically, it is ubiquitylated) which releases the replicative DNA polymerase and, together with damaged DNA, attracts a translesion polymerase specific to that type of damage. Once the damaged DNA is bypassed, the covalent modification of the clamp is removed, the translesion polymerase dissociates, and the replicative polymerase is brought back into play. DNA RECOMBINATION Segments of DNA can rearrange their location – within a chromosome – from one chromosome to another Such recombination is involved in many biological processes – Repair of DNA – Segregation of chromosomes during meiosis – Enhancement of genetic diversity In sexually reproducing organisms, recombination and mutations are two driving forces of evolution – Recombination of co-infecting viral genomes may enhance virulence and provide resistance to antivirals THREE CLASSES OF RECOMBINATION Homologous/general recombination – Exchange between two DNAs that share an extended region of similar sequence Site-specific recombination – Exchange only at a particular sequence DNA transposition – “jumping genes” – short DNAs that can move from one chromosome to another Transposons, first discovered in the 1940s by Cornell alum and Nobel Prize-winner Barbara McClintock, are abundant components of genomes – they make up half of human DNA – and have the ability to hop and replicate selfishly in the genome. Some transposons contain their own genes that code for enzymes called transposase proteins, which cut and paste genetic material from one chromosomal location to another. THREE FUNCTIONS OF HOMOLOGOUS RECOMBINATION 1) Assists in DNA repair 2) Links sister chromosomes to properly segregate them between self and daughter cells 3) Source of DNA exchange and therefore genetic diversity DOUBLE STRAND BREAKS CAN BE REPAIRED BY TWO MECHANISMS Two ways to repair double strand breaks: (A) NONHOMOLOGOUS END JOINING alters the original DNA sequence when repairing a broken chromosome. The initial degradation of the broken DNA ends is important because the nucleotides at the site of the initial break are often damaged and cannot be ligated. Nonhomologous end joining usually takes place when cells have not yet duplicated their DNA. (B) HOMOLOGOUS RECOMBINATION is more difficult to accomplish but restores the original DNA sequence. It typically takes place after the DNA has been duplicated (when a duplex template is available) but before the cell has divided. Details of the homologous recombination pathway are presented in the next slides NON-HOMOLOGOUS END JOINING (NHEJ) 18, 495–506 (2017) Schematic of DNA double-strand breaks (DSBs) and their repair by non-homologous end joining (NHEJ). The Ku70–Ku80 protein heterodimer binds to DSBs and improves their subsequent binding by the NHEJ polymerase, nuclease and ligase complexes. These enzymes can act on DSBs in any order to resect and add nucleotides. Multiple rounds of resection and addition are possible, and nuclease and polymerase activities at each of the two DNA ends seem to be independent. Microhomology between the two DNA ends, which is either already present (dashed boxes) or newly created when the polymerases add nucleotides in a templateindependent manner, is often used to guide end joining. The process is error-prone and can result in diverse DNA sequences at the repair junction (bottom). However, NHEJ is also capable of joining two DNA ends without nucleotide loss from either DNA end and without any addition. Nucleotide additions are depicted in green lower case. HOMOLOGOUS RECOMBINATION IN BACTERIA Replication fork encounters damage in template strand Replication fork collapses due to creation of a double-strand break – leaves two parts of a strand to be processed 5’-ending strand is degraded 3’-single-stranded extension is bound by a recombinase, pairs with complementary sequence in intact duplex DNA, “invades” the duplex à creates branched structure of three strands Branch moves to create X-like structure known as “Holliday junction” or “Holliday intermediate” A nuclease and ligase restore the structure of the replication fork HOMOLOGOUS RECOMBINATION can flawlessly repair double strand breaks in DNA This is the preferred method for repairing DNA double-strand breaks that arise shortly after the DNA has been replicated, while the daughter DNA molecules are still held close together. In general, homologous recombination can be regarded as a flexible series of reactions, with the exact pathway differing from one case to the next. For example, the length of the repair “patch” can vary considerably depending on the extent of 5′ processing and new DNA synthesis, indicated in green. HOMOLOGOUS RECOMBINATION can rescue broken DNA replication forks When a moving replication fork encounters a singlestrand break, it will collapse, but can be repaired by homologous recombination. The process uses many of the same reactions shown in the previous slide and proceeds through the same basic steps. Green strands represent the new DNA synthesis that takes place after the replication fork has broken. This pathway allows the fork to move past the site that was nicked on the original template by using the undamaged duplex as a template to synthesize DNA. SUMMARY In this lecture, we learned: Several mechanisms exist to correct mismatches and other changes in DNA During DNA repair, the information encoded in the parent strand can be used to make corrections in the daughter strand Homologous recombination involves swapping of regions of DNA with similar sequences