DNA damage and repair

Question 1

DNA replication properties:

Answer 1

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

Question 2

3 steps of replication in E. coli

Answer 2

initiation (at oriC)
elongation (bi-irectionally from oriC)
termination (Ter region)

Question 3

DNA pol III properties at replication fork

Answer 3

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

Question 4

elongation at replication fork?

Answer 4

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

Question 5

3 pols needed for DNA synthesis

Answer 5

DNA pol I
DNA pol III
Primase

Question 6

where does initiation occur?

Answer 6

OriC
has 13mer DNA repeats
then 9-mer repeats a bit downstream

Question 7

how does initiation start?

Answer 7

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

Question 8

continuation of initiation?

Answer 8

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

Question 9

termination

Answer 9

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

Question 10

problems encountered in replication?

Answer 10

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)

Question 11

replication restart

Answer 11

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

Question 12

another problem replication fork encounters

Answer 12

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…

Question 13

how can repair proteins cause breaks?

Answer 13

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)

Question 14

linear DNA formation at Tus?

Answer 14

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

Question 15

SOS response in E. coli?

Answer 15

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

Question 16

SOS response genetic structure?

Answer 16

Regulon (group of genes under control of same regulator)
controlled by LexA

contains different genes with different function located on different parts of chromosome

Question 17

gene functions in SOS regulon?

Answer 17

-HR
-nucleotide excision repair
-polymerase
-cell division
-SOS regulator (lexA)
-Toxic - halt cell growth

Question 18

SOS system function when no damage?

Answer 18

no dna damage
= LexA proteins bind specific consencus sequence at promoters of the SOS regulon genes (the LexA box)
Lex A = transcriptional repressor

Question 19

LexA repressive function?

Answer 19

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

Question 20

different sensitivities to LexA repression?

Answer 20

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

Question 21

SOS function when cell experiences stress

Answer 21

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)

Question 22

SOS response when damage is not repaired

Answer 22

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

Question 23

roles of proteins expressed in SOS response

Answer 23

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))

Question 24

Early SOS response?

Answer 24

EARLY:
tries to repair DNA while protecting MAXIMUM dna integrity
high fidelity synthesis to faithfully replicate damage
high fidelity polymerase

Question 25

middle SOS response? (still somewhat early)

Answer 25

HR and NER proteins expressed to repair damage
these early proteins need to repair ASAP as faithfully as possible

Question 26

later SOS response

Answer 26

UH OH
SOS response still active
need to stop cell cycle so next gen doesn’t inherit damage

SOS expresses proteins that:
halt cell cycle/prevent division
toxic proteins that momentarily halt cell growth

cell grows slower and stops dividing

Question 27

SOS last resort:

Answer 27

all attempts to repair DNA faithfully have failed
SOS respsonse expresses low fidelity error prone polymerases
carry out translation synthesis
LAST RESORT that come with cost
can produce mutations

but some mutations better than having broken genome and definitely dying

Question 28

consequences of SOS response?

Answer 28

HR - genome rearrangement
important for DNA repair but the rearrangement can happen
trying to repair with other similar DNA sequences at different points on chromo:
can cause
genome deletion, duplication, inversion - can change gene dosage

increased mutation rate - error prone polymerases cause mutations through error making
mostly neutral or deleterious (sadge)
if deleterious cell dies or grows slower (disadvantage in its progeny :()

so best to control levels of these proteins
also don’t want these happening under normal conditions as DNA damage and cell cycle halting are not in cell’s advantage
need to grow and divide to colonise environment and increase chances of survival and large progeny

Question 29

SOS response side effect advantage:

Answer 29

in presence of selective agent in environment (e.g. anitmicrobial)
increases cell survival beyond just DNA repair
SOS induced mutations can give new alleles which can lead to opportunities for adaptation
gives more chance of evolving antimicrobial resistance
gives it advantage in surviving and replicating in presence of this selective agent

Question 30

Break join recombination model?

Answer 30

2 dna molecules (black and blue)

break in both

ends ligate to other end of WRONG molecule

formation of new genetic material
(blue-black molecule and vice versa)

both molecules broken, no new DNA synthesis)

not really correct at all as new genetic materia from H recombination usually features old and new DNA, and presence of heteroduplex DNA

Question 31

Copy choice recombination model?

Answer 31

both DNA molecules being replicated
at some point templates are exchanged
results in formation of new genetic material
original DNA has not been broken
new genetic material consists entirely of newly synthesised DNA

Question 32

Break copy recombination model?

Answer 32

one of dsDNA molecules has been broken

uses other molecule as template for repair

in this case:
one of original dsDNA molecules has been broken
new genetic material partially newly synthesised