Lecture 9 DNA Repair 2 NER Flashcards
3 types of excision repair
Mismatch repair MMR - removes newly synthesised strand after replication
base excision repair BER - removes a single damaged base. Has DNA glycosylases that recognise specific types of damage
Nucleotide excision repair NER - removes damage as a short oligonucleotide. Deals with a large range of structurally unrelated and helix-distorting DNA lesions. Excised strand removed by helicase and resulting gap filled by a DNA pol.
Net is a versatile system that can repair Cyclo butane pyrimidine dimers (CPD) the (6-4) photoproduct, chemical adducts such as O6-METHYLGUANINE DNA crosslinks etc. Hence humans don’t have DNA photolyase they use NER to deal with pyrimidine dimers.
Ner was discovered in 1964 in studies on processing of UV damage in bacteria. Genes that gave resistance to damage named Uvr (ultraviolet resistance)
NER in bacteria
In bacteria incisions are made 12 or 13 nucleotides apart. A 12 nt strand is removed with a monoadduct and a 13 nt strand by a diadduct; the 5’ cut site is fixed at the 8th phosphodiester bond 5’ of the damaged site whereas the 3’ cut site is at the 4th or 5th bond 3’ of the damaged site. The gap is filled by DNA pol l
Repair of interstrand crosslink
They prevent DNA strands opening for rep and transcript.
Repair usually requires nucleotide excision repair and homologous recombination although there are actually multiple complex pathways for repair.
DNA pol that can bypass lesions can help this process with risk of making mistakes.
Translesion bypass DNA polymerases
E. Coli has 2 DNA polymerases capable of bypassing lesions by inverting any nucleotide at damaged site. They are called Pol lV (DinB) and Pol V (UmuD’2C) both are tightly regulated so that this type of DNA synthesis only occurs where cells are so damaged that managing to replicate and transcribe takes precedence over risk of mutation generation. Sometimes called error prone DNA polymerases. So E. Coli has 5 DNA polymerases 3 high fidelity (l ll lll) and 2 ‘sloppy’ IV and V
Bacterial NER proteins
Protein/ amino no./properties
UvrA, 940, binds UV irradiated dsDNA can dimerise and complex with UvrB has 2 ATP binding sites and 2 zinc fingers
UvrB, 673, Binds Uvra2 5’3’ DNA helicase activity as a UVRA2B2 complex has 1 ATP binding site
UvrC, 610, interacts with UvrB-DNA complex has 2 endonuclease active sites makes 5’ and 3’ incisions
UvrD, 720, 3’5’ DNA helicase binds ATP and DNA releases (unwinds) oligonucleotides containing damaged bases
Mfd, 1148, specificity for transcribed strand repair by UvrABC displaced RNA pol interacts with UvrA
PolA, 928, DNA pol l fills ssDNA gap left by release of excised oligonucleotide
LigA, 671, seals nick (reforms diester bond) completing repair process
GGR global genomic repair / global NER
Random search for lesions in DNA distinct from transcription coupled repair. UvrABC aka (A)BC exinuclease: excision nuclease (because UvrA is not involved at cutting stage). Expression of UvrA B and D is induced as part of the SOS response ( group of genes involved in DNA repair whose expression is stimulated following DNA damage)
During global NER a DNA lesion is recognised by a UvrA and UvrB complex usually A2B or A2B2. UvrC is recruited and cleaves the damaged strand on both sides of the lesion. UvrD (DNA helicase ll) then displaces both UvrC and the 12-13 nucleotide section containing the damaged base or bases. Resulting gap filled by DNA pol l which displaces UvrB and DNA ligase seals nick at 3’ end of repair patch.
A cascade of recognition by UvrABC gives high specificity for damage recognition. UvrA is a molecular matchmaker- it loads UvrB onto damaged DNA but does not take part in the remainder of the reaction. Base pairing of DNA near the lesion is disrupted and DNA bent by UvrB a beta hairpin inserted between the 2 strands of DNA locks the protein in place ready for UvrC to arrive. UvrC contains two distinct endonucleases that make the 5’ and 3’ incisions flanking the damaged nucleotide(s)
Transcription - coupled repair (TCR)
Describes preferential repair of transcribed strands. DNA lesions that impede DNA and RNA polymerase can kill cells by a) interfering with DNA replication in actively growing and dividing cells b) blocking transcription and thus depriving the cell of essential proteins. Cells have a system that targets DNA repair enzymes to transcriptionally active genes.
In bacteria RNA pol stalled at a lesion is recognised by enzyme called Mfd (mutation frequency decline) aka transcription repair coupling factor (TRCF)
Mutation frequency decline (MFD)
Complex phenomenon discovered 1956 by Evelyn Witkin. Holding bacterial cells in poor medium inhibited protein synthesis temporarily and gave cells time to repair damage from UV light preferentially on the transcribed strand. Cells defective in Mfd gene do not exhibit this mutation frequency decline and a link to nucleotide excision repair was uncovered. The protein responsible Mfd (TRCF) wasn’t discovered until 1990
MFD
RNA pol stalled at a lesion obscured recognition of the damaged DNA by the NER factors UvrA and UvrB this preventing repair. Stalled RNA pol is recognised by Mfd ( via an RID - RNA polymerase interacting domain, binds to a region on beta subunit of RNAP) which uses its translocate activity to track along the DNA and eject both RNAP and transcript from the DNA. From then on the pathway is identical to GGR carried out by UvrABC.
TCR results in damage repaired faster than it would be in non-transcribed regions that are repaired by GGR pathway by UvrABC alone..
Basic steps of bacterial and human NER are essentially the same although a larger oligonucleotide (27-30nt) is excised in the eukaryotic system, reflecting the larger no. Of proteins and complexity of the process in higher organisms
