DNA damage and repair Flashcards
DNA replication properties:
semi conservative
need to separate duplex strands (one strand kept in new molecule and other one newly synthesised)
Uses dNTPs to synthesise new strand
DNA pol catalyses synthesis of new strands by addition of dNTPs
3 steps of replication in E. coli
initiation (at oriC)
elongation (bi-irectionally from oriC)
termination (Ter region)
DNA pol III properties at replication fork
performed by DNA pol III
main replicative polymerase
subunits:
alpha - 5’-3’ polymerisation
epsilon subunit - proofreading exonuclease
theta subunit - core assembly and stability (not really known??)
gamma complex - 5 subunits - loads and ubloads polymerase onto DNA template via clamp
beta subunit - sliding clamp - tethers pol to DNA
stays just behind it to ensure its stays in right direction - doesn’t go back
tau subunit - connnects two pol III a subunits to create dimeric polymerase unit (two strands being copied at once)
needs primer to begin synthesis
small RNA primer produced by DNA primase provides free 3’ OH for DNA pol III to extend from
5’-3’ direction
elongation at replication fork?
you know how leading and lagging are synthesised
(multiple primers and fragments for lagging)
DNA pol III acts as dimer
so lagging strand loops around so both pol move in same direction (diagram)
RNA primers removed by repair DNA pol I
removes primer
simultaneously adds new nucleotides from 3’ end on new strand to replace primer
ligase ligates end of new strand to its start to form loop
same for lagging strand but multiple times to link Okazaki fragments
3 pols needed for DNA synthesis
DNA pol I
DNA pol III
Primase
where does initiation occur?
OriC
has 13mer DNA repeats
then 9-mer repeats a bit downstream
how does initiation start?
DnaA protein loaded with ATP
this allows it to bind to 9mer repeats
this binding causes DnaA proteins to bind to 13mer repeats too on same strand (see diagram)
this opens the DNA a bit at 13mer repeat region at OriC
allows for DnaB to bind - the main replicative helicase (a hexamer circle)
can open its circle and enclose around ss DNA at oriC
continuation of initiation?
after DnaB helicase loading onto DNA
opens DNA helix bi directionally
SSB can bind to newly opened ss regions to prevent refolding into double helix
gyrase (a topoisomerase) can help deal with the supercoiling changes happening due to replication
termination
other side of chromo to oriC
Terminus regions (Ter)
Tus protein binds some of these sites
bound Tus can stop replication fork coming from other side
Tus binds a Ter site
one fork from one side reaches and stops now waiting for other fork
other fork arrives from other side and converges
two forks join (diagram)
replisome removed from DNA
couple different proteins (esp RecQ and TopoIII) help to get rid of replication proteins that still persist
so each fork replicates about half of chromosome
problems encountered in replication?
ss gap forms in parental DNA ahead of replication fork
if fork not there yet can remove damaged nucleotides and and resynthesise
if replisome reaches this gap before repaired
transformed into ds end (major cause of ds break, BAD potential loss)
small problem became big problem
can cause loss of genetic info (mutations jinkies!!)
RecBCD processes this ds end as it would in HR, strand invasion with RecA and holliday junction
RuvABC binds and translocates holliday junction away from D loop
(see diiagram)
this causes a 3 way juntction (as only one ds end instead of two in break)
recognised by replication restart proteins (PriA most important)
replication restart
RecBCDprocesses ds break from replisome catching up to ss gap
then RuvABC processing:
Half holliday junction cut and rearranged so it resembles replication fork
PriA recognises 3 way junction and loads onto it
opens DNA and loads accessory proteins onto it (PriB, DnaT, DnaC)
once DnaC loaded, DnaB (main replicative helicase) can be loaded onto opened DNA
once DnaB has opened DNA more, can reload rest of replisome
another problem replication fork encounters
replication fork reversal
replisome deflects or template damage block/stalls fork
obstacles that deflect replisome can be:
strong binding proteins to DNA (e.g. a repressor)
3 way junction (fork from parental strands and replicating strands)
becomes 4 way holliday junction
(deflection causes replisome to walk back - causing annealing of replicating strands and formation of 4 way)
replication reversal dependent on RuvAB
then RuvC cuts and repairs by HR…
how can repair proteins cause breaks?
RecA:
binds ss region between discntinuous okazaki fragments on lagging strand near replication fork
complementarity between ss part of parental strand and other parental strand
-> RecA invades
forms holliday junction
RuvA will bind this
then RuvC can cut it
this causes DS breaks
BUT
RecBCD can recognise this
fixes it (you know how 😊😊)
can also get repair by PriA pathway (idk how in this case just be aware ig)
linear DNA formation at Tus?
Ter site binds Tus protein
fork stops when hit Tus
then if new Replication forks have formed at OriC can reach site where two older forks are before they’ve managed to converge
“over-replication” - rare
forms linear DNA - drawing diagram of it not hard
SOS response in E. coli?
refulatory system for survivinig DNA damage (stress response to DNA damage)
balance between replicating faithfully and repairing breaks in time
more severe the breaks - less faithful more emergency replication
response PROPORTIONAL to extent of damage
as components of DNA repair may not be faithful or cause damage themselves
musn’t pass on damage to next generation
halts cell cycle until repaired
SOS response genetic structure?
Regulon (group of genes under control of same regulator)
controlled by LexA
contains different genes with different function located on different parts of chromosome
gene functions in SOS regulon?
-HR
-nucleotide excision repair
-polymerase
-cell division
-SOS regulator (lexA)
-Toxic - halt cell growth
SOS system function when no damage?
no dna damage
= LexA proteins bind specific consencus sequence at promoters of the SOS regulon genes (the LexA box)
Lex A = transcriptional repressor
LexA repressive function?
LexA dimer binds LexA box
interferes with action of RNA pol - inhibiting expression of SOS genes
most genes in regulon have basal expression level but this is much lower than levels in sos response
LexA box near or in DNA pol binding sites
-10 recognition site recognised by Pol
overlaps with the LexA box there
different sensitivities to LexA repression?
sequence of LexA box next to gene is important
more similar to consensus sequence - stronger binding = more difficult for LexA to leave the box
so SOS genes expressed earlier in response have LexA box LESS similar to consensus sequence for weaker LexA binding and easier de-repression
number of LexA box and location of LexA boxes in promoter also important to sensitivity
SOS function when cell experiences stress
stress
dna break
RecBCD processes the ds break
loads RecA to form RecA nucleoprotein filament
active RecA np filament acts on LexA as a coprotease
induces self cleavege of cytoplasmic LexA
LexA conc decreases
now less LexA present to bind LexA boxes
so now can’t bind boxes with sequence most different from consensus sequence
this allows access of RNA pol onto promoter of early SOS genes (so they can now be expressed more)
SOS response when damage is not repaired
RecA ss np filament remains
lowers cytoplasmic concentration of LexA even more
leads to derepressio of more genes later into SOS response
the longer the damage goes unfixed - teh less LexA - the derepression of more and more SOS genes later and later into response
roles of proteins expressed in SOS response
main:
LexA
RecA
feedback loops:
DNA repaired
so now no longer RecA as ss np filament
no more cleavage of LexA
SOS response repressed
other members of regulon further regulate it by loops:
some proteins stabilise the RecA ss np filament
others destabilise it
SOS response highly regulated as it encodes for DNA replication methods
(HR (faithful)
Nt excision repair (faithful)
translation sysnthesis (depending on conditions can be faithful/unfaithful))
Early SOS response?
EARLY:
tries to repair DNA while protecting MAXIMUM dna integrity
high fidelity synthesis to faithfully replicate damage
high fidelity polymerase
