plant molecular and cellular biology...8/20/2008 pmcb lecture 4: g. peter 2 learning objectives 1....

8/20/2008 1

Plant Molecular and Cellular BiologyLecture 2: Mechanisms of DNA

Repair

Gary Peter

8/20/2008 PMCB Lecture 4: G. Peter 2

Learning Objectives1. Explain the role for

DNA repair in high-fidelity DNA replication

2. Describe the structures and functions of enzymes responsible for DNA repair

3. Explain their roles in DNA repair

4. Explain the in vitroapplications of DNA repair enzymes for recombinant DNA methods


Mutations and DNA Repair

Mutations occur if the DNA is not repairedThe damage that occurs is usually to one-two bases on one strand, thus the opposite strand provides the template for insertion of the correct base


Overview of DNA Repair

DNA repair is essential for survival5,000 purine bases are lost per cell per day from thermal fluctuationsIncreases fidelity of DNA replication by ~100 fold over DNA polymerases


Common Steps in Repair Pathways

Recognition of the altered baseFacilitated by structure of double helix

Removal of alterationSynthesis of correct nucleotide

Facilitated by the structure of double helix & sister chromatidsSome exceptions occur here: direct reversal, nonhomologous end joining, error prone DNA repair


Common Forms of Damage to Bases in DNA

The glycosidic bond of nucleotides is prone to acid-catalyzed hydrolysis to form abasic sites

~10,000 abasic sites per human cell per day (10,000/3x109 = 1 in 3 x 105 bases)

Detection and repair of damaged bases is facilitated by the structure of DNA


Direct Reversal

Two types of altered bases can be directly reversed

O6-alkylguanine adducts are directly removed by O6-alkylguanine transferasesPyrimidine dimers induced by UV light are directly reversed by photolyases


Direct Reversal of Damaged Bases: O6-alkylguanine Transferase

In most organisms, O6-alkylguanine adducts can be repaired by alkylguanine transferases (AGT)AGTs transfer the alkyl group from the altered guanine base in the dsDNA to a reactive cysteine group in an irreversible reaction

Angew. Chem. Int. Ed. 2003, 42, 2946 – 2974

The EMBO Journal Vol. 19, pp. 1719-1730, 2000

http://emboj.oupjournals.org/content/vol19/issue7/images/large/e070903.jpeg


Direct Reversal of Damaged Bases: Photolyases

UV-induced pyrimidine dimers (cyclobutane pyrimidine dimers) can be repaired by nucleotide excision (mammals) or by direct reversal of the damage by photolyases in plants, lower eukaryotes, and bacteria

Photolyases contain a chromophore which absorbs light and transfer of the excitation energy to the FAD cofator for electron transfer to the pyrimidine dimer to initiate the reversion

Current Opinion in Chemical Biology 2001, 5:491–498


Base Excision Repair: DNA Glycosylases

Damage to DNA bases from deamination, oxidation, demethylation and alkylation is mainly repaired by base-excision repairMultiple different DNA glycosylases recognize specific damaged bases and initiate their repair by base excisionRepair is then subsequently completed by a number of different enzymes

Angew. Chem. Int. Ed. 2003, 42, 2946 – 2974


DNA Glycosylase: Uracil GlycosylaseDeaminated cytosine residues are uracilsUracil DNA glycosylase recognizes the U:G mismatch and excises the base from the DNA strand by the hydrolysis of the N-glycosidic bond between the base and the sugar phosphate leaving the backbone intact and producing an abasic siteAP endonuclease hydrolyzes the phosphodiester bond 5’ to the abasic site to generate a nick Removal of the abasic site occurs by the AP lyase activity of DNA polymerase βDNA polymerase β also adds the single new C and DNA ligase seals the nick


Uracil DNA GlycosylaseUracil DNA glycosylase is the most active of the four DNA glycosylases that show activity towards deaminated C (uracil)

105 faster than other DNA glycosylases

UDG interacts with the replication machinery and appears to clear the genome of U immediately after replicationAll DNA glycosylases use a common nucleotide flipping mechanism

Target nucleotide is extruded out of the dsDNA into the active site

UDG and SMUG1 are the only glycosylases that act on both ssDNA and dsDNA

Angew. Chem. Int. Ed. 2003, 42, 2946 – 2974


In Vitro Use of UDGPreventing carryover contamination in PCR

Incorporate dUTP by Taq into PCR productsTreat with UDG to produce abasic DNA which will no longer amplify


Nucleotide Excision RepairNER is the repair pathway that removes a variety of bulky adducts from DNA

Pyrimidine dimers –UVEnvironmental mutagensChemotheraputic agents

Two main pathways Global genome repair (GGR) for untranscribed regions, the bulk of the genomeTranscription-coupled repair (TCR)

This broad substrate specificity is remarkable when compared with the specificity in base excision repairThe efficiency of repair of the different adducts can vary several orders of magnitude and generally correlates with the degree of helical distortion caused by the alteration

Note simple mismatches or bubbles are not substrates, so distortion of the DNA backbone is insufficient


Nucleotide Excision Repair: E. coliThis pathway requires the uvrABCencoded exinuclease, a helicase encoded by uvrD, and DNA polymerase I.

UvrA is both an ATPase and a DNA-binding protein (it contains Zn-finger motifs). It functions as a dimer and it recognizes and binds to damaged DNA. The function of UvrA is to lead UvrB to the site of damage.UvrB is an endonuclease and an ATPase, although the ATPase activity is cryptic and is only revealed when it is complexed with UvrA.UvrC then binds to UvrB. This complex nicks the DNA on either side of the lesion or damage. UvrC nicks DNA about 7 nucleotides on the 5' side of the damage; UvrB nicks DNA about 4 nucleotides on the 3' side of the damage.The UvrD helicase binds to this region and unwinds it. By so doing, it displaces the short single strand carrying the site of the damage. In total a region of 12-13 nucleotides is removed. This region is then repaired by DNA polymerase I and DNA ligase.


Mechanism of NER:GGR in Eukaryotes

The steps are Recognition of damaged residuesBubble formation Dual excision of the damaged DNA strand 5’ and 3’ to the lesion, Release of the 24-32 bp oligonucleotide containing the damage, Repair synthesis Ligation of the gap

Angew. Chem. Int. Ed. 2003, 42, 2946 – 2974


Mechanism of Global Genome Repair

The core NER reaction has been reconstituted in vitro and about 30 proteins contribute to NER

Damage recognition & bubble formation

XPC-hHR23B, TFIIH (9 subunits includes helicase), XPA, RPA (trimeric SSB), XPG

Incision & release of oligonucleotide

Endonucleases XPG and ERCC1-XPF

DNA fill-inPol δ and pol ε, sliding clamp PCNA, pentameric clamp loader RFC

LigationDNA ligase I

Angew. Chem. Int. Ed. 2003, 42, 2946 – 2974


Mechanism of Transcription-coupled Repair

TCR was discovered from observations that lesions which block RNA polymerases are repaired more rapidly in transcribed parts of the genome

Initial damage is recognized by stalled RNA polymeraseAll the proteins involved in GGR except XPC-hHR23B are required for TCR.TCR requires additional proteins including CSA & CSB


Mismatch RepairThe MMR system increases the fidelity of DNA replication

Eliminates base mismatches, nucleotide deletions and insertions introduced by DNA polymerases

MMR enzymes recognize 1-2 chemically altered bases in a mispair or loopMMR is conserved from bacteria to humans, but with some notable differences

In prokaryotes, the newly synthesized DNA strand is recognized before it is methylated

MMR enzymes recognize the hemi-methylated DNA and act only on the unmethylated strand

Eukaryotes, don’t have hemi-methylated DNA after DNA replication and the recognition mechanism is unknown

One model shows that the MMR machinery is coupled to the replication apparatus


Mismatch Repair: E. coliIn E. coli, the initiator is the MutS homodimer, which binds to mismatches and insertion/deletion loops

Binding of MutS to the alteration is the rate limiting step for repair

Upon binding, MutS triggers ATP-dependent assembly of the repairosome, during which MutS moves away from the mismatch and MutL homodimer is recruitedMutL acts as a bridging factor for MutH, which nicks the newly synthesized strand 5’ of the nonmethylated GATC/(GAmeTC)This nick serves as an entry point for helicase II and one of a few exonucleases (Exo VII, Rec J, or Exo I) which degrade the nicked strand past the mismatch. The ssDNA is protected by SSB The gap is filled in by DNA pol III DNA ligase repairs the gap


Structure of E. coli MutSThe two identical subunits interact with the mismatch as a functional dimer

One interacts with the mismatchOne interacts with the parent DNA

DNA is kinked by ~60o

at the site of the mismatch

Angew. Chem. Int. Ed. 2003, 42, 2946 – 2974


Mismatch Repair: EukaryotesNo mutH similar genes are in eukaryotes consistent with a different strand recognition mechanismThe MSH6-MSH2 (MutSα) heterodimer recognizes the mismatch to initiate repair

MutSα binds to single mismatches and to small/insertions and deletion loopsMutSβ only binds to insertion/deletion loops of various sizesATP hydrolysis drives the threading of the DNA through the MutSαMutSα or MutSβ dimers trigger assembly of the MMR machinery

MLH1/PMS2 (MutLα) and PCNA are recruited

PCNA is the processivity factor in replication

Exo 1 is involved with MMR, but no other exonucleases or helicases have been identified Angew. Chem. Int. Ed. 2003, 42, 2946 – 2974


Double Stranded BreaksDouble stranded breaks are induced by ionizing radiation, oxidizing agents, replication errors and specific metabolic products cellsUnrepaired lesions would quickly lead to the breakdown of chromosomes into smaller fragments


Repair of Double Stranded BreaksTwo independent pathways

Homologous end-joiningGeneral recombination mechanisms transfer the sequence information from the intact chromosome to the site of the ds-breakThis HR pathway is important for meiosis, repair of interstrand crosslinksThe HR pathway is important in S and G2 phases, during which the sister chromatid is available

Nonhomologous end-joiningBroken ends are juxtaposed and rejoined by DNA ligation with the loss of 1-2 nucleotides at the endsNHEJ pathway appears important for quiescent or terminally differentiated cells and in G1NHEJ is required for immune diversity and telomere maintenance


Repair of DSBs by Homologous Repair

HR is a highly complex pathway

End recognition and processing

Processing the ends of the break by RAD52 and exonucleases generate 3’ single-stranded tails

Angew. Chem. Int. Ed. 2003, 42, 2946 – 2974


Strand Invasion: Homologous Pairing and Strand Exchange

Strand invasionRPA binds to the 3’ overhangsRad51, a recA homologue, assembles onto the single strands to form nucleoprotein filamentsNucleoprotein filaments search for homology in the donor template

Rad54 facilitates this process by interacting with Rad51 and stimulates the strand exchange reactionRad54 is a Swi2/Snf2 family member involved with chromatin remodeling


Repair Synthesis and Branch Migration

Repair synthesis is mediated by DNA polymerasesIn E. coli RuvA and RuvB proteins mediate branch migration and RuvC catalyzes resolution of Holliday junctions

RuvA-RuvB form an active complex

Angew. Chem. Int. Ed. 2003, 42, 2946 – 2974

http://www.sdsc.edu/journals/mbb/abcomplex.html


Nonhomologous End JoiningKu70-Ku80 heterodimer binds to the ends at the ds-break and recruits DNA-PK (DNA protein kinase)Bridging of the two ends requires the Rad50/Mre11/Nba1 and Lig4/XRCC4 complexes which both interact with the Ku proteins


DNA End Recognition by Ku70/80

Ku70 & Ku80 share limited sequence identity, but show similar folding patterns

They bind to the DNA as a dimer

DNA binds through the ring and the Ku proteins do not directly contact the bases only the phosphate backbone


Inducible DNA Repair Systems: SOS Response in E. coli

The SOS repair system is induced in response to major damage to the bacterial DNA or in response to agents which inhibit DNA replication. The system is a complex one with over 20 genes involved. Two of these are the important regulator genes: lexA and recA.LexA is a repressor that regulates the expression of all of the other SOS repair genes, including recA. It also regulates its own synthesis (i.e. it is autoregulatory). Normally, LexA blocks expression of the SOS repair genes.The RecA protein is a multifunctional protein with ATPase and ssDNA binding activities. When bound by ssDNA, it is also a co-protease. Damage or severe stress to the cell generates ssDNA which activates this co-protease activity. The RecA co-protease activity upon binding to LexA stimulates the co-protease activity of the LexA protein. As a result, LexA is no longer able to block transcription and the SOS repair genes are thereby induced and expressed.


Inducible DNA Repair Systems: SOS Response in E. coli

Among the genes that are induced are uvrABC and Dand also umuC and umuD. UmuD is cleaved by the RecA coprotease activity and the truncated protein, UmuD', in association with UmuC forms DNA polymerase V. Pol V requires the β and γ subunit of Pol III for optimal activity. DNA synthesis by Pol V is error-prone.Error prone DNA synthesis can be harmful to individual cells, but must be advantageous for the population


Summary

Multiple repair systems have evolved to protect the genome from mutation These systems involve multiple proteins with multiple different functionsEach of these systems must identify the few bases that need to be repaired in the genome

plant molecular and cellular biology...8/20/2008 pmcb lecture 4: g. peter 2 learning objectives 1....

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