Sarah Dermier Flashcards
what are replication forks?
sites of DNA synthesis inside cells
which direction is DNA synthesised?
5’ to 3’
what are the three key polymerases in eukaryotic cells and how do they differ?
polymerase alpha, delta and epsilon
alpha and delta have low processivity (i.e. stop synthesis frequently) while epsilon has high
delta and epsilon has 3’ exonuclease activity so can also cut DNA
what is the MCM protein complex aka helicase?
unwinds DNA in advance of the replication fork i.e. runs ahead of the polymerase to make sure they can bind
involves multiple proteins e.g. GINS, cdc45 which form complex with MCM to aid DNA unwinding
what does polymerase epsilon do?
low error rate and high processivity
the main processing polymerase of the leading strand and needs alpha polymerase (primase) to prime the DNA initially
what does proliferating cell nuclear antigen (PCNA) do?
acts like a clamp holding polymerase epsilon and delta down to enable high processivity
what key proteins does leading strand synthesis involve?
helicase
polymerase epsilon
PCNA
what are the key proteins in lagging strand synthesis?
replication protein A (RPA)
polymerase alpha
replication factor C (RFC)
polymerase delta
Fen1 (aka MF1) and RNase H
what does replication protein A (RPA) do?
binds single stranded DNA template to keep it single stranded (no enzymatic activity)
what does polymerase alpha do?
associated with a seperate enzyme (primase) which synthesises the RNA primer that starts DNA synthesis
polyA then takes over and starts DNA synthesis making the DNA primer (only like 20bp hence no issue with polyA low processivity and fidelity)
polyA and primase displace RPA by making the RNA and DNA primers
what does replication factor C (RFC) do?
binds the 3’ end of the DNA primer displacing polyA and primase and initiating the switch to polymerase delta
this step called polymerase switching
also loads PCNA onto the DNA
what is polymerase delta?
takes over lagging strand synthesis from polymerase alpha
what is the purpose of RNase1 and Fen1?
once polymerase delta doing synthesis theres a bunch of fragments (cause lagging strand) and you need to make this one continuous strand and also get rid of RNA primers
so when poly delta hits RNA primer for next fragment it runs through a bit pushing up RNA primer and a bit of DNA (strand displacement) allowing exonucleases RNase1 and Fen1 to come in and cleave off RNA primer and DNA respectively
once exonucleases RNase1 and Fen1 have chopped off the displaced strands, what is the final step of lagging strand synthesis?
DNA ligase comes in and ligates the fragments together to give continuous strand
why does only polymerase delta have strand displacement activity?
its (pretty much) the only one doing lagging strand synthesis i.e. leading strand has no okazaki fragments
what makes polymerase delta have high processivity?
PCNA
what proteins do polymerase A interact with?
mcm10 (part of helicase)
polymerase delta
what proteins does polymerase delta interact with?
PCNA
polymerase A
what proteins does polymerase epsilon interact with?
cdc45, GINS (both part of helicase)
PCNA
what is the replisome?
replication fork + all the associated proteins + DNA
where does replication start?
starts at origins of replication (ori)
eukaryotic chromosomes have multiple origin sites
when and where are replication forks initiated?
initiation of replication occurs at different sites at different times during S phase
nearby sites tend to be activated together and regions of high gene density get replicated first
replication forks from each ori outwards till it meets a fork from the next ori
what are the two kinds of chromatin in eukaryotic cells?
euchromatin - more open and actively transcribed (replicated first cause more gene density)
heterochromatin - more densely packed and closed (replicated later cause more gene depleted)
outline yeast origins of DNA replication?
first isolated from yeast saccharomyces cerevisiae
ori in yeast called autonomously replicating sequence (ARS)
100-200bp; functional/essential regions of origin can be identified using mutations and seeing how that mutation affects origin function
outline origins of DNA replication in higher eukaryotes?
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
what is ORC?
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
outline the assembly of the replisome?
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
during assembly of the replisome, how does loading of helicases occur?
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
how do (and what) kinases control replication?
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
what are the phases of the cell cycle?
G1 - growth
S - DNA synthesis
G2 - growth and prep for mitosis
M - mitosis (cell division)
outline control of replication in the context of the cell cycle?
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
how does licensing and unlicensing work?
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
how is cdc6 and cdt1 activity controlled in yeast?
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)
what proteins form the licensing system?
ORC
cdc6
cdt1
mcm2-7
summarise replication initiation regulation (licensing system) in yeast?
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)
summarise replication initiation regulation in higher eukaryotes?
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
how do checkpoint proteins provide a safety net during replication initiation?
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
what are some applications/implications of
proteins involved in replication initiation?
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
describe the diversity/differences between DNA polymerases?
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
what is the Y family of DNA polymerases?
has high error rate and can do translesion synthesis (TLS) meaning can synthesise through damaged sites
why/how do diff DNA polymerases have different fidelities?
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)
what things initially make DNA synthesis so accurate?
free energy is different for abnormal base pairs
DNA polymerases have narrow active sites that reduce the likelihood of an incorrect nucleotide being added
what three options does DNA polymerase have when it incorporates the wrong base?
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
what is DNA proofreading?
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
why do correctly matched basepairs have less likelihood of moving into exonuclease site?
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
how is the incorporation of ribonucleotides prevented and why do they occur?
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
outline DNA damage?
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
how does DNA damage effect DNA replication?
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
discuss the switching of polymerases when encountering DNA damage?
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
discuss how different DNA polymerases bypass different types of DNA damage?
polymerase eta bypasses UV lesions e.g. TT dimers
polymerase kappa bypasses abasic sited
polymerase zeta bypasses ribonucleotides
what is structurally and functionally different about Y family DNA polymerases?
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
how does the differing active site of TLS polymerases allow them to synthesise through lesions?
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
what are the implications of DNA errors for both humans and b acteria?
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
what is xeroderma pigmentosum?
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
what is cytosine deamination?
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
how does formation of abasic sites occur and what can it lead to?
damaged bases (e.g. deamination), inappropriate bases (e.g. uracil), normal purines getting lost
can lead to translesion synthesis and incorporation of incorrect bases
how does oxidative damage cause DNA lesions?
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
how is 8-oxoguanine repaired?
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
how does OGG1 remove 8-oxoG?
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
what is the difference between short and long patch base excision repair?
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
why is the 8-oxoG repair system so important?
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
what causes single strand breaks?
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
what causes ds breaks and what do they result in?
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
what are the four repair pathways for ds breaks?
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)
discuss non-homologous end joining (NHEJ)?
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
what can go wrong with non-homologous end joining?
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.
what is the DNA mismatch repair system?
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%
what are the applications and implications of DNA repair mechanisms?
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
