Sarah Dermier Flashcards

Question

outline origins of DNA replication in higher eukaryotes?

Answer 1

30-100kb apart, best studied in drosophila and human more complex than yeast ori - no defined DNA sequence starting replication although A-T rich other factors determining ori include chromatin structure (high gene density regions first), nucleosome position, transcriptional activity

Answer 2

origin recognition complex; binds to and remains at origin sites throughout cell cycle highly conserved with core group of six proteins; Orc1-5, cdc6 forming ring around DNA Orc proteins have ATPase activity i.e. DNA binding is ATP-dependent

Answer 3

ORC loaded onto DNA after mitosis Cdc6 proteins attach to the ORC, MCM-helicase protein complex recruited after G1 phase (during S phase) other DNA synthesis proteins recruited to form pre-initiation complex (PIC) MCM-protein complex unwinds DNA (two of these going both directions outwards), priming occurs and then DNA synthesis begins

Answer 4

cd6c6 comes in and completes orc complex, after which helicase can bind once the first helicase binds cdt1 and cdc6 dissociate and bind again in different orientation this recruits the second helicase which will go opposite direction

Answer 5

at enzymatic level this process works by kinases which phosphorylate proteins and activate them once helicase loaded DDK phosphorylates helicases after this Sld recruited and then S-CDK once in S phase and phosphorylates polymerase Then MCM10 comes back in triggering helicase activity leading to formation of replication bubble THUS replication is a complex sequential process requiring energy from kinase activity to start

Answer 6

G1 - growth S - DNA synthesis G2 - growth and prep for mitosis M - mitosis (cell division)

Answer 7

replication occurs only once per cell cycle in the S phase replication origins are "licensed" for replication during the M and G1 phases - this means MCM protein complexes are assembled

Answer 8

MCM helicase proteins move away from origin site for DNA replication and are not loaded back onto ORC complexes until after cells have divided (after mitosis) activity of cdc6 and cdt1 proteins downregulated at end of G1 phase (unlicensing); this done cause we only want to replicate things once cdc6 (subunit of ORC complex) and cdt1 are binding partners of M protein so activity of these controls licensing/unlicensing

Answer 9

CDK (cyclin-dependent kinase) protein prevents re-replication by acting on cdc6; phos it after which it degraded also phos cdt1 (--> export from nucleus) and ORC (MCM protein exported from nucleus) cdc6 synthesis also transcriptionally regulated (occurs in late mitosis, G1)

Answer 10

ORC cdc6 cdt1 mcm2-7

Answer 11

during mitosis: orc inhibited, cdc6 degraded, cdt1 and mcm2-7 exported; CDKs being expressed and regulating these during G1: all proteins in licensing system active and CDKs not expressed during S phase: replication initiation has started; CDKs regulating like in mitosis phase again (inhibit, degrade, export) so the process is tightly regulated and controlled (and needs to be)

Answer 12

during mitosis: orc and cdc6 inhibited, cdt1 inhibited by geminin protein; regulated by geminin and CDKs during G1: all licensing system proteins active during S phase: orc ubiquitylated and degraded, cdc6 prob degraded, cdt1 degraded; geminin regulating noone really knows what going on w mcm again process very tightly controlled

Answer 13

overexpressing cdt1, or mutants affecting ORC, cdc6 or MCM can cause re-replication this activates checkpoint kinases that detect abnormal DNA structures these activate pathways stopping DNA synthesis at 'wrong' rep forks, stopping cell cycle and inducing apoptosis if checkpoints compromised this can result in cancer

Answer 14

meier-gorlin syndrome inherited human disease associated with ORC protein mutations cdt1 or cdc6 over-expressed in some tumours correlating with unusual chromosome rep some anticancer treatments target CDKs

Answer 15

many more pol than just alpha, delta, epsilon all with diff roles some start DNA synth (alpha), some do lagging strand synth (delta), some do leading strand synth (epsilon), some have proofreading ability (epsilon, delta), some involved in DNA repair (delta, gamma), some do translesion DNA synthesis can be grouped into families based on protein sequence

Answer 16

has high error rate and can do translesion synthesis (TLS) meaning can synthesise through damaged sites

Answer 17

delta, epsilon have low error rate (high fidelity) alpha has average error rate TLS polymerases (Y family) such as polymerase eta have high error rate (low fid)

Answer 18

free energy is different for abnormal base pairs DNA polymerases have narrow active sites that reduce the likelihood of an incorrect nucleotide being added

Answer 19

dissociate (an external exonuc may come in and fix) DNA pol has its own exonuc activity (proof reading) so can cut out wrong base wrong base actually gets incorporated, might get repaired, if not = mutation

Answer 20

involves 3'-5' exonuclease activity mismatched base pairs are poor substrates for addition of new nt (processivity drops and so pol pauses); pause allows DNA to unwind 3' end of new DNA where mismatch is which moves into exonuclease reaction site and is cleaved off DNA synthesis then resumes

Answer 21

correctly matched basepairs are good substrates for DNA synthesis so less time to move into site (high processivity) incorrectly matched aren't so DNA pol pauses allowing unwinding and moving of 3' end of new DNA into exonuclease site --> cleavage

Answer 22

dNTPs and rNTPs identical apart from additional -OH group making rNTPs in DNA more likely to cause strand breakage polymerase epsilon incorporates ~1 rNTP per 1,250 dNTPs (quite frequent) so mechanism needed to remove ribonuclease enzyme scans along DNA and cleaves out any incorporated rNTPs

Answer 23

over 50,000 lesions occur each day in human cell DNA e.g. oxidative damage (guanine to 8-oxoguanine) loss of a base (abasic sites) dimerisation of adjacent pyrimidines

Answer 24

absence of valid template base causes replicative DNA pol to stall allowing replication complex to disassemble/pol dissociate class Y translesion (TLS) DNA polymerases can then take over which are specific to target lesions - can synthesise across incorrect base

Answer 25

when DNA pol dissociates there is switching event and Y family TLS pol comes in to synth through lesion pol switching occurs due to ubiquitination of PCNA which causes replisome to disassemble; Y family pol Rev1 then comes in and recruits other TLS polymerase; details of this bit still unclear TLS pols error prone so only synth small region (<200bp)and there is second switching event

Answer 26

polymerase eta bypasses UV lesions e.g. TT dimers polymerase kappa bypasses abasic sited polymerase zeta bypasses ribonucleotides

Answer 27

no detectable sequence similarity with other DNA polymerases similar structure as other DNA pols but with additional domain holding DNA substrate in place have low fidelity with non-damaged DNA templates due to no proofreading exonuc but this also how they can bypass damaged DNA site

Answer 28

TLS DNA polymerases have a much more open active site allowing DNA damaged sites with non-standard basepairs to fit in there if a replicative polymerase hits a TT site it can't fit this in its active site

Answer 29

DNA breakages can lead to cell death incorporating the wrong base can lead to mutations can result in disease in humans (e.g. xeroderma pigmentosum), or antibiotic resistance in bacteria

Answer 30

first TLS pol (eta) was discovered in this disease; autosomal recessive, causes extreme sunlight sensitivity, cancer develops at sites of UV exposure so they have mutation in pol eta which synth through UV damage sites thus end up with lots of mutations

Answer 31

if cytosine gets deaminated (N replaced w oxygen) it becomes uracil; an RNA base we don't want cause changes pairing (U-A as opposed to C-G) C can also be methylated; if methyl group deaminated it becomes thymine paired w G when DNA replicates half the daughter DNA is mutant i.e. C deamination can cause wrong bases to be incorporated into newly synth DNA

Answer 32

damaged bases (e.g. deamination), inappropriate bases (e.g. uracil), normal purines getting lost can lead to translesion synthesis and incorporation of incorrect bases

Answer 33

G can get turned to 8-oxoguanine due to ROS; this still can pair w C but extra oxygen can flip base around allowing pairing w A as if its T; this called hoogsten pairing so next replication cycle on daughter strand G-C and one A-T; this lesion cannot be bypassed so must be repaired

Answer 34

oxyguanine glycosylase (OGG1) can remove 8-oxoG; repair mechanism then adds G if A already paired adenine glycosylase (MUTYH) can cut it out then cycle repeats and 8-oxoG can be removed by OGG1 glycosylases break bond between DNA backbone and sugar

Answer 35

scans along dsDNA strands flipping out each G as it goes along; only 8-oxoG fits well in its active site --> gets cleaved so OGG1 can distinguish between G and 8-oxoG as only the latter can fit in active site allowing it to break glycosidic bond i.e. OGG1 high specificity

Answer 36

when OGG1 cleaves out 8-oxoG we still need to fix abasic site; APE1 (AP nuclease) comes in and makes ss cleavage; pol beta comes in and synth correct base; DNA ligase repairs break; fixed thats for only one base (short); replicative pol and PCNA can come in and synth more than one base (2-20) creating flap; FEN1 cleaves DNA flap; DNA ligase comes in and seals backbone making perfect dsDNA; long patch base excision repair so difference is one base getting repaired vs 2-20

Answer 37

defects in MutYH associated w colorectal cancer; families with this defect experience GC-->TA transversions mutations in MutYH reduce enzyme affinity for 8-oxoG mutations in MutYH and OGG1 in mice increase chance of tumours and increase amount of 8-oxoG in DNA

Answer 38

reaction of deoxyribose w ROS during base excision repair action of topoisomerase that controls DNA supercoiling presence of ribonucleotides in DNA repair mechanisms for all the above can leave ss breaks sometimes; this can lead to ds breaks which are much worse

Answer 39

ss breaks in a template strand get passed by a replication fork can also occur due to radiation or oxygen radicals result in genome instability which can lead to cancer if cell checkpoints fail

Answer 40

homologous recombination (HR) using sister chromatid as template non-homologous end-joining (NHEJ) 'alternative' NHEJ (a-EJ) single-strand annealing (SSA; leads to deletions between repeat sequences)

Answer 41

protein Ku70/80 scans along DNA; ring structure so can only bind DNA unless theres a ds break cause (their entry point) if clean break can be ligated together; usually ends been degraded and bp lost so need to fill these up Ku70/80 will recruit a bunch of other factors which fill in, trim back (to generate blunt ends) and ligate the ds break ends back together

Answer 42

if more than one ds break DNA ends may get joined together that were not previously joined; results in translocations (chromosome in wrong place) processing may delete important base pairs; if section where break occurred important for protein function the deletion can make it non-functional defects in DSB repair system lead to immunodeficiency, extreme sensitivity to ionising radiation etc.

Answer 43

even w normal template DNA pol can incorporate wrong bases into DNA --> mismatch MSH2/MSH6 protein complex recognises mismatch; Exo1 catalyses removal of a short patch of DNA containing wrong base; DNA repair synthesis by pol delta replaces this DNA mismatch repair system reduces mismatch frequency by about 99%

Answer 44

defects in repair pathways increase mutation frequency leading to disease inhibitors of OGG1 can SUPPRESS cancer cell proliferation (cause cancer cells have high rates of 8-oxoG) NHEJ and/or HR essential for successful CRISPR/Cas9 gene editing cause creates ds breaks