SCGI W6-8 Flashcards
why do mutations in NHEJ give rise to SCID/CID
DNA DSBs are physiologically induced during immune system development - V(D)J-recombination and class switching recombination (CSR)
V(D)J recombination = process of cutting/pasting DNA within the B and T cell receptor loci to create huge amounts of diversity within the antigen binding part of the receptor
RAG1/2 recognise specific sequences within the B and T cell receptor loci that allow DSB induction
DSBs in the BCR and TCR are predominantly repaired by NHEJ - unrepaired DSBs trigger activation of p53-dependent apoptosis
patients with mutations in NHEK genes often present with severe combined immunodeficiency due to an inability to repair DSBs induced during V(D)J recombination
microcephaly and the NHEJ genes
DNA DSBs can naturally occur during S-phase due to replication fork collapse
increased proliferation increases the chances that replication forks will collapse into DSBs
rapidly dividing neuro-progenitor cells will generate more DSBs that slower replicating cells and such have a higher dependence on DSB repair pathways for their repair
if DSBs aren’t repaired properly in the VZ by HR as the neuro-progenitor cells move into the SVZ and start to differentiate and come out of cycle - NHEJ will be required to repair these breaks - inability ti repair these DSB = trigger p53-dependent apoptosis and neuronal loss
mutations in HR genes and immunodeficiency
loss of ATM - components of the MRN complex/RNF168 don’t result in a major problem with V(D)J recombination
A-T/A-TLC/NBS/NBSLD patients have recurrent spontaneous chromosomal translocations involving immune receptor genes
ATM plays a role in stabilising post-cleavage complexes of coding ends
A-T/NBS/RIDDLE patients experience a reduction in Ab production (IgG/IgA) - inactive defect in CSR
CSR mechanism where B cells switch from IgM to other Ig isotypes
CSR also involves the generation and repair of DNA DSBs
loss/mutation of the ATM-MRN pathways -> CSR dependent DSBs repaired by Alt-EJ -> internal deletions within BCR switch regions + non-productive CSR events - reduction of IgG/A/D/E production
HR gene mutations and microcephaly
during development the neuro-progenitor cells rapidly expand to prodcue a sufficient number of cells to allow the brain to develop properly
genetic mutations that slow the cell cycle of neuro-progenitor cells reduces the total pool of cells required for brain development - results in small brain
severity of the cell cycle defect correlates with the severity of the reduction in head size
DNA DSBs occur naturally during S-phase due to replication fork collapse
increased proliferation inc. chance of replication fork collapse
rapidly dividing neuro-progenitor cells generate more DSBs than slower replicating cells - higher dependence on HR for repair
mutations in ATM and MRE11 and cerebellar degeneration
brain development in A-T/A-TLD patients = normal, cerebellum degenerates once the brain is fully developed
A-T/A-TLD/NBS/NBSLD cells exhibit many common cellular defects
unknown as to why the cerebellum deteriorates
chromatin packaging and signalling
how the chromatin is packaged effects gene expression
signalling enabled the expression/switching off of genes
remodelling of the chromatin allows for altered expression
the nucleosome
nucleosome = core histones: tetramer of H3/4 and dimer of H2A/B above/below
146 bp of DNA wrap around the nucleosome in a left handed superhelix - tethered to the the nucleosome by H1 (linker histone)
the flexible N terminal tail = the point of signalling
the histone tail
signalling occurs via post-translational modifications
- allows for complexes to open/close a region of DNA for expression
flexible and unstructured
post translational modifications
acetylation
methylation
phosphorylation
ADP-ribosylation
ubiquitination
acetylation
occurs on the e- amino group of specific lysins at the amino-terminal end of all CHs
H4 - 4 sites - least-K16 -> K12 & K8 -> K5-most
H3 - 5 sites - least-K14 -> K23 & K27 -> K9 & K18-most
acetyl CoA -> CoA via acetyltransferases [HATs]
acetyl lysine -> lysine via deacetylases [HDACs]
block action of HDACs via Na Butyrate/Na Valproate leads to hyperacetylated histones
RNA polymerase II holoenzyme
turn a gene off temporarily/permanently
interaction and combination of post translational modifications that occur both on the histone and on the DNA
RNA polymerase II holoenzyme
turn a gene off temporarily/permanently
interaction and combination of post translational modifications that occur both on the histone and on the DNA
acetylation effect on chromatin
acetylation increases accessibility of TFs to DNA by opening up chromatin
chromatin closed - transcriptionally inactive, low levels of acetylation
chromatin open - transcriptionally active, high levels of acetylation
euchromatin = acetylated
heterochromatin = hypoacetylated
methylation
occurs on specific lysine residues
methyltransferases
1/2/3 methyl groups added to each e-amino group
methylation occurs on CHs histones H3 and H4 ONLY and on H1
- H3 methylated at lysine 4/9/27
- H4 methylated at lysine 20
- H1 methylated at lysine 26
methylation effect on chromatin and DNA
methylation at specific lysine residues on H3 signals DIFFERENT complexes
active chromatin - high levels of acetylation, high levels of H3K4 methylation, DNA unmethylated
heterochromatin - methylation at H3K9 signals HP1 to bind, spreading of HP1 coated nucleosomes silences heterochromatin
inactive chromatin - low levels of acetylation, high levels of H3K27 methylation, DNA methylated
phosphorylation
in vivo proteins can be phosphorylated at 2 types of amino side chain
- O-phosphate linkage [phosphate for a OH group] e.g. serine threonine tyrosine
- N-phosphate linkage e.g. lysines histidines arginines
phosphorylation: protein kinase - ATP -> ADP
removal of phosphate: protein phosphatase
histone phosphorylation
binds to the dyad axis where the DNA wraps around the nucleosome + crosses over
H1
- 3 serine residues [S17/S169/S185]
- 2 threonine residues [T134/T151]
H3
- 2 serine residues [S10/S28]
position of phosphorylation alters the stability of H1 binding to the nucleosome
when are histones phosphorylated
highest level of histone phosphorylation observed at mitosis - H3 and H1
also occurs on
- H3 as cells are stimulated from quiescence to growth by GFs
- only present on a small number of genes (e.g. immediate early genes)
different combinations of histone modifications and their outcomes
condensation caused by high levels of
- H3K9 methylation
- H3K27 methylation
- H3S10/S28 phosphorylation
opening cased by high levels of
- H3K9 acetylation
- H3S10 phosphorylation
- H3K27 acetylation
same post translational modification on a residue, the adjacent modification is the riving force for the change in transcriptional activity
enzyme families that modify histone tails
AcetylCoA - acetylation (lysine)
S-adenosyl methionine - methylation (lysine, arginine)
ATP - phosphorylation (serine)
the epigenetic code
changes to DNA and its associated proteins that can alter gene expression without altering the DNA sequence
- altering DNA methylation
- histone modification
single-nucleosome code/multi-nucleosome code
histone acetylation
acetylation
- intermediary metabolism
de-acetylation
- environmental stress
- intermediary metabolism
- signalling pathways
- therapeutic inhibitors
epigenetic modifications: histone modifications
a combination of different molecules can attach to the tails of histone proteins
alter the activity of the DNA wrapped around them
epigenetic modifications: DNA methylation
methyl marks added to certain DNA bases repress gene activity
epigenetic modifications: micro RNAs
RNA molecules that are not translated into protein but still functional
methylation of cytosine in DNA
5th DNA base
occurs on CpG islands
- CpG dinocleotides are palindromic
difference in heterochromatin and euchromatin DNA methylation and histone modifications
euchromatin
- high histone acetylation
- low DNA methylation
- H3-K4 methylation
heterochromatin
- low histone acetylation
- dense DNA methylation
- H3-K9 methylation
maintenance of methylation
DNA methyltransferases (Dnmts) are essential
maintenance: Dnmt1 - puts on the mirror methylation onto the daughter strand
Dnmt3A/B responsible for new methylation
methylation of DNA from a zygote to an adult
totipotent - devoid of DNA methylation
pluripotent - some regions are methylated (turn off genes that code for extra-embryonic tissues)
multipotent - acquiring more DNA methylation (turn off different lineages)
unipotent - specific gene expression profile (majority of genome silenced by DNA methylation)
pluripotent cells most CpG islands will be unmethylated, unipotnet cells most CpG islands will be methylated
de-methylation of the paternal pronucleus
occurs at the 1 cell stage embryo
in embryogenesis
male genome demethylation occurs as the gametes fuse
female genome demethylation takes longer and is complete by the morula stage
genomic imprinting
some genes are expressed only from the maternal genome and some are only from the paternal genome
estimated that around 80 genes are imprinted - found on several different chromosomes
epigenetic genomic imprinting
modification of specific genes during gametogenesis so that only the paternal or maternal allele is expressed after fertilisation - “parent of origin” gene expression
affects the expression but NOT transmission of alleles
2 gene copies present
1 gene copy active = functional haploidy
genomic imprint - the unequal expression of the maternal and paternal alleles of a gene
epigenetic genomic imprinting: Igf2 and H19
insulin growth factor 2 (Igf2) paternally expressed
H19 (a non-coding RNA) maternally expressed
regulated by a cis-acting, differentially methylated region (DMR) = an imprinting control region (ICR)
epigenetic genomic imprinting: Igf2 and H19 mechanisms
paternal DMRs methylated = H19 OFF and Igf2 ON
- DMR1 is a silencer inactivated by methylation
- DMR2 is an enhancer that is activated by methylation
maternal CMRs hypo-methylated = H19 ON and Igf2 OFF
- CTCF binds DMR
- downstream enhancer engaged for H19 expression
- DMR1 silencer of Igf2 active
- DMR2 enhancer of Igf2 is inactive
genomic imprinting in mammals
parent of origin gene expression imprints must be correctly set in the gametes every generation
failure to reset correctly leads to developmental disorders in the next generation
prader-willi syndrome
mostly sporatic
deletion at 15 q11-q13
obesity, short, small hands/feet, unusual facial features, mild mental retardation
compulsive overeaters
angelman syndrome
speech impairment
movement/balance disorder
behavioural uniqueness
excitable personality
maternal chromosome deletion UBE 3A
roles of DNA methylation
transcriptional silencing
protecting the genome from transposition
genomic imprinting
X inactivation
tissue specific gene expression
complexity of human disease
the following contribute to the disease phenotype
genetic mutation/alteration
environmental factors
imprinting and epigenetics
lifestyle factors
inherited diseases
2 types
- multigenic diseases
- monogenic diseases
multigenic diseases
mutations in multiple genes
arise from less severe mutations in multiple genes
any one particular mutation may not result in a disease phenotype: only when they are combined does the disease phenotype occur
established by analysing function interactions between candidate genes - epistasis
monogenic diseases
mutations in single gene
e.g. cystic fibrosis (CFTR)/ataxia telangiectasia (A-T/ATM)
caused by more severe mutations in single genes
tend to be rare due to selective pressure against deleterious mutations
may be caused by recessive mutations and therefore escape selective pressure
monogenic inherited diseases and the DNA damage response
lost of different sources of DNA damage
- IR/X-rays/chemotherapy
- MMS/EMS/chemo
- MMC/cisplatin
- metabolism/chemo
- CPT
developmental diseases focus around
- brain development
- growth development
- premature ageing
monogenic inherited diseases and the DNA damage response examples
fanconi anaemia (FA)
seckel syndrome (SS)
Ataxia-Telagiectasia (A-T)
Ataxia-Telagiectasia-like disease (ATLD)
Nijemgen breakage syndrome (NBS)
Nijemgen breakage syndrome-like disorder (NBSLD)
werner syndrome (WS)
bloom syndrome (BS)
fanconi anaemia
bone marrow failure
skeletal defects
genome instability (chromosomal aberrations)
myeloid leukaemia
seckel syndrome
growth retardation
dwarfism
microcephaly
mental retardation
genome instability (chromosomal aberrations)
ataxia-telangiectasia
genome instability (chromosomal breakage)
T/B cell leukaemia/lymphoma
ataxia-telangiectasia-like disease
neurodegeneration
ataxia
genome instability (chromosomal breakage)
nijmegen breakage syndrome
microcephaly
growth retardation
genome instability (chromosomal breakage)
T/B cell lymphoma
nijmegen breakage syndrome-like disorder
microcephaly
growth retardation
mental retardation
genome instability (chromosomal breakage)
werner syndrome
premature ageing
growth retardation
genome instability (chromosomal aberrations)
various cancers
bloom syndrome
premature ageing
growth retardation
genome instability (chromosomal aberrations)
leukaemia/lymphoma
genetic linkage
non-random association of markers as they are passed from parent to offspring
linkage studies tracks the inheritance of various markers
- several 1000 restriction enzymes
- several million SNPs
analysing genetic linkage
- obtain samples from at least 2 generations of a family with affected and unaffected individuals
- genotype DNA using markers
- analyse each markers for linkage to the disease - analyse the regions conserved between affected individuals and carriers
- evaluate all genes in the region
- identify the causative mutation
identifying where a disease gene lies
principle: if 2 sequences lie close together they are likely to be inherited together
close-together sequences are less likely to be separated during inheritance
identifying regions of homology from linked markers
principle: a mutation shared by affected individuals through common descent will be surrounded by shared alleles at nearby loci
genetic markers for linkage e.g.s
restriction sites (older + simplistic)
SNPs (newer + powerful)
microsatellites - short tandem repeats
restriction enzyme markers
single base pair mutations can alter the ability of restriction enzymes to cut DNA at specific sequences
can trace inheritance of the gene by following the inheritance of a particular pattern of restriction fragments
SNPs
single nucleotide polymorphisims: single base pair variations
scattered randomly throughout genome (approx 100-300bp intervals)
can occur in coding/non-coding regions
may be linked with disease phenotypes
act as markers of variation and inheritance
SNP arrays
cheap and relatively powerful
robust: much less laborious and less error prone than using other markers
with online bioinformatics database - have a good idea of the genetic location of millions of SNPs - enormous coverage
commercial libraries of SNPs are easily available
data can give insight on inheritance mapping/mutation mapping
GWAS
genome wide association study
can correlate disease or susceptibility with SNPs
used to discover new gene variants associated with a particular phenotype/disease
comparative genome arrays
aCGH used to analyse whole-genome changes
exome sequencing
label the DNA and capture the exons
read them
give the mutations in the coding sequence of the genes
good for looking at only 1 or 2 patients
pull out 100 mutations in 9000 different genes
whole genome sequencing
finding the functional importance is difficult unless you have lots of patients/ a good idea of where to start
deciphering developmental disorders
DDD aims to find out if using new genetic technologies can help doctors understand why patients get developmental disorders
- systemic application of the latest microarray and sequencing methods
deciphering developmental disorders
DDD aims to find out if using new genetic technologies can help doctors understand why patients get developmental disorders
- systemic application of the latest microarray and sequencing methods
100000 genomes project
project focuses on patients with a rare disease and their families + patients with common cancers
ATR mutations identified as the cause of SCKL1
autozygosity/homozygosity mapping
- used to examine mutations in small populations
- mutations likely to be caused by homozygous changes originating from descent by inbreeding (autozygosity)
- areas of homozygosity near to markers are examined and checked in parents
-regions of homozygosity indicate areas of interest in which the candidate genes lay
- can use traditional sequencing or SNPs
- identified region surrounding 3q22.1-24 as an area of interest - process of elimination identified ATR within this
LOD scores
LOD () score = a statistical test used for linkage analysis
compares the probability of obtaining the test data if 2 loci are linked to the likelihood of observing the same data by chance
high LOD = traits are closely linked -> inherited together
low LOD = traits aren’t closely linked
based on assumptions of genetic distance
post-translational modifications
writer - adds the modifications
editor - removes the modifications
cause biochemical changes in protein shape/charge/size
which cause functional changes in localisation/enzyme activity/complex formation/stability
read by a reader
post-translational modifications e.g. phosphorylation
writer = kinase ATP->ADP
editor = phosphatase
reader = phospho-peptide binding domain
types of post-translational modifications
phosphorylation - S/T/Y
acetylation - K
methylation - K/R
glycosylation - N/R/S/T/Y…
hydroxylation - P/K
SUMOylation - K
ubiquitylation - K
small modifier proteins
share the same underlying structural fold
have different amino acid sequences so exhibit different surface charge
e.g. ubiquitin (1UBQ)/SUMO1 (1ASR)/SUMO (1WM3)
small modifier cycles
E1 - activating enzyme
- energy dependent step
- addition of Ub
E2 - conjugating enzyme
- Ub passed from E1 to E2 active site
E3 - ubiquitin ligase
- forms a complex with E2
- provides specificity
reverse = deubiquitinating enzyme (DUB)
the ubiquitin cascade
3 enzyme cascade facilitates complexity and specificity within the ubiquitin pathway
2 E1s, 40 E2s, 700 E3s
complexity of ubiquitin modifications
mono-ubiquitination
multi mono-ubiquitination: multiple Ubs at different sites
poly-ubiquitination: formed on lysine residues on Ub can form up to 7 different chains
- homotypic chains
- heterotypic chains
- branched chains
complexity of ubiquitin modifications: chains
K6
K11
K27
K29
K33
K48
K63
M1 (linear)
complexity of ubiquitin modifications: chain shape and function
different chain types have different shapes which have different functions
DNA damage response:
K6 chains - BRCA1-BARD1 - unknown function
K11 chains - RNF8-Ube2S - DNA damage response
K48 chains - RNF8/4 - protein degradation & protein signalling
K63 chains - RNF8-Ubc13/RNF168-Ubc13 - DNA damage complex formation
ubiquitin binding domains
proteins that read the the Ub signal contain a Ub binding domain (UBD)
some proteins contain more than 1 UBD - the orientation of the UBDs allows them to selectively bind to specific ub-ub linkages
greater than 20 different families of UBDs
proteins containing multiple domains with multiple functions
the orientation and proximity of the dual UIM domains in RAP80 make this region a specific binder for K63-linked Ub chains
the orientation and location of multiple UBDs in the same protein can determine specificity for polyubiquitin Ub-chain binding - RNF168 gives specificity for K63-linked Ub-chains
ubiquitination mechanisms
conjugation of a target protein by Ub
requires the activity of 3 enzymes, which provides
- specificity
- flexibility
- complexity
1000s of proteins are modified by UB not all are then degraded
proteins that have multiple Ub binding domains may be able to specifically recognise poly-ubiquitination chains over mono-ubiquitination events
ubiquitination in the DNA damage response to double stranded DNA breaks
E3 Ub ligases
Ub in the DNA damage response
DNA damage occurs in the context of chromatin
nucleosomes are both modified and act as scaffolds for proteins recruited to sites of DNA damage
Ub in the DNA damage response: phosphorylation
phosphorylation events initiate the response leading to the recruitment of Ub writer RNF8
Ub in the DNA damage response: RNF8
RNF8 activity initiates the Ub signal K63-Ub chains on histone H1
RNF8 activity also labels JMJD2A with K48-Ub chians marking it for proteasomal degradation
Ub in the DNA damage response: RNF168
RNF168 reads the RNF8 K63-Ub signal via ubiquitin interacting motifs (UIMs)
RNF168 propagates the Ub signal (K63-chains, H2A K13/15 mono-Ub and K27-chains)
Ub in the DNA damage response: recruitment of factors
recruitment of repair decision factors (53BP1/BRCA1) by specific Ub signals
53BP1 recognises H2A-K13/15Ub and H4-K20me2
BRCA1 brought in through the Rap80/abraxas complex
Ub in the DNA damage response: choice of repair pathway
HR or NHEJ
how the BRCA1 and 53BP1 complexes talk to each other determines the pathway repair choice
decision is also regulated by the cell cycle
resection is a critical step
Ub in the DNA damage response: G1
G1 - 53BP1 complex is dominant
53BP1
- reads the H2A-K13/15-monoUb and H4-K20me2
- blocks the recruitment of resection enzymes
- blocks the retention of the BRCA1 complex
no resection therefore promotion of NHEJ
Ub in the DNA damage response: S and G2
dual UIM domains in RAP80 read the K63-Ub chain mark
BRCA1 write H2A-K125/127/129-Ub mark which is read by SMARCAD1
SMARCAD1 remodels nucleosomes and moves 53BP1 away from the damage site
BRCA1 promotes recruitment of resection enzymes (CtlP/BLM/Exo1)
promotes resection and HR
Ub in the DNA damage response: importance
loss of control in these Ub modification pathways is associated with clinical disease
mutations in RNF168 are associated with RIDDLE syndrome
mutations in BRCA1 are associated with an increased risk of breast and ovarian cancer
BRCA1 -ve tumours show high levels of genomic instability - due to loss of HR
initially responsive to PARP inhibitors - without HR the cells can’t process PARPi-mediated DNA damage
rapidly develop chemoresistance - further loss of 53BP1 in the tumour leads to restoration of HR
SUMO
small modifier cycles
SUMO conjugation primarily occurs at large hydrophobic K-x-D/E motifs
3 conjugatable isoforms
- mono-SUMOylation: SUMO1/2/3
- multi mono-SUMOylation: SUMO1/2/3
- poly-SUMOylation: SUMO2/3
SUMO readers (SUMO interacting motifs (SIM))
SIM
when they occur in tandem allows for the recognition of poly-SUMOylation
SUMO in the DNA damage response
SUMOylation is involved in
- recruitment of complexes
- activating BRCA1 ligase
- regulating chromatin state
SUMO E3 ligases: PIAS1 and PIAS4 = writers
SUMO and Ub crosstalk in the DNA
RNF4 contributes to clearing factors from damage sites to allow repair to proceed
1. MDC1 SUMOylation by SUMO E3 ligase PIAS4
2. RNF4’s STUbL activity marks MDC1 with K48-Ub chains and targets MDC1 for degradation
3. removal of MDC1 allows the DNA damage response to proceed
ubiquitin and SUMO an=re dynamic modifications - Editors
DeUbiquitinating or SENP enzyme
Ub and SUMO editors in the DNA damage response
SENP2 limits SUMO chains on MDC1 to regulate timing of MDC1-ubiquitination and removal from sites of DNA damage
H2A-Ub DUBs
- USP3 inhibits recruitment of RNF168, 53BP1, and RAP80
- USP44 over expression prevents 53BP1 foci forming
- USP48 removes H2A-K125/K127/K129-Ub to control resection lengths and prevent over-resection
POH1 limits 53BP1 spread
Ub and SUMO editors in the DNA damage response
SENP2 limits SUMO chains on MDC1 to regulate timing of MDC1-ubiquitination and removal from sites of DNA damage
H2A-Ub DUBs
- USP3 inhibits recruitment of RNF168, 53BP1, and RAP80
- USP44 over expression prevents 53BP1 foci forming
- USP48 removes H2A-K125/K127/K129-Ub to control resection lengths and prevent over-resection
POH1 limits 53BP1 spread
relationship btw viruses and the DDR
all viruses cause the cell to activate the DDR following infection
due to
- the cell recognises the viral DNA as its own broken DNA triggering the response
- it forms part of a normal anti-viral response
- stress caused by the virus invading the host cell triggers the DDR
- the virus intentionally activates the DDR as it will aid viral replication in some way
activation of the DDR may therefore be beneficial and/or detrimental to the virus
DNA viruses
HPV small double stranded circular genome linked to cervical cancer
adenovirus dsDNA virus with linear genome (about 35Kb) causes mild respiratory diseases
kaposi’s sarcoma associated herpesvirus (KSHV) a gamma herpes virus responsible for Kaposi’s sarcoma which is the most common cancer in sub-saharan africa
HPV
over 100 different serotypes
infect different epithelial sites
very strong linked to cervical cancers and now to cancers of the oro-pharynx
HPV genome circular ds DNA
DDR proteins affected by and co-localising with HPV E1 and E2
HVP and the DDR
HPV activates the DDR mainly through the action of E1 and E2 proteins although the presence of viral DNA may contribute to this - seen as an activation of ATM signalling
numerous DDR proteins are seen at HPV replication centres such as Rad51/RPA/BRCA1/ATRIP/TOPBP1
E6 and E7 encourage cellular proliferation but also impinge on the DDR
E7 tends to inhibit the DDR generally whilst E6 causes the degradation of p53 causes degradation of MGMT and p300 in some viral serotypes - also interacts with XRCC1 - also inhibition and activation of the FA pathway
HPV E1 and E2 proteins
E1 induces activation of ATM causing cell cycle arrest in S and G2 activation of ATM is probably due to direct damage to cellular DNA
E1 probably activates ATR seen as phosphorylated Chk1
E2 activates the ATM pathway seen as phosphorylated Chk2
HPV E6 and E7 proteins
E6 inhibits SSB repair reduces ATR levels and generally inhibits the ability of the cell to repair UV induced damage - impinges positively and negatively on the FA pathway
E7 impairs HR repair increasing the persistence of gamma-H2AX and RAD51 foci - has been reported to activate ATM - E7 induces ATR pathway activation as well as inducing replication stress
expression of E6 and E7 results in stalled replication forks and DSBs
activation of ATR/Chk1 and E2F by HPV31 results in an increase in RRM2 which increases the pool of dNTPs facilitating viral replication
inhibition of ATR/Chk1 results in inhibition of viral replication
Adenovirus (Ad)
small non-enveloped DAN tumour virus
linear double-stranded genome
oncogenicity dependent on virus serotype, dose and host immune status
60+ different serotypes divided into 6 species
Adenovirus (Ad) causes
inhibits the DDR seen as a lack of phosphorylation by degrading important proteins such as Mre11 and BLM
failure to inactivate host DDR causes an inability to replicate as the over-sized genomes cannot fit into the viral capsid
Adenovirus (Ad) and degradation
Ad E1B-55K targets DNA damage response proteins for degradation: p53/MRE11/Rad50/NBS1
- Ad5 uses Ub ligase Cul5 to degrade p53
- Ad12 uses Ub ligase Cul2 to degrade p53
TopBP1 degradation by Ad12 is E1B-54K independent
Ad viral replication centres
Ad E2 proteins mark viral replication centres
RPA is re-localised to viral replication centres during Ad infection
ATRIP and RPA32 co-localise at viral replication centres
alternative activation of the MRN complex
Ad infection
virus genome
E1B-55K/E4-ORF3 inhibit MRN
global DDR phosphorylation
inhibiting viral replication
Ad and the MRN complex
RN complex prevents virus genome replication through a mechanism that doesn’t activate global ATM signalling/require MRE11 exonuclease activity
inactivation of MRN enables virus genome replication and triggers downstream global DDR signalling, inactivation is through either protein degradation or localisation to aggresomes or E4orf3 tracks
initial replication of Ad genomes is semi-conservative similar to cellular DNA - shown that MRN specifically senses early replicating viral genomes
ATM has an role in MRN binding in preventing the viral genome replication - MRN binding near viral replication origins physically prevents the progression of viral DN replication
EBV and genomic instability
role of pathogenesis of burkitt’s lymphoma
exogenous/endogenous DNA damage
inactivation of DNA damage-response pathways
inactivation of apoptosis/mitotic checkpoint pathways
leading to cancer
Kaposi’s sarcoma-assocaited herpesvirus (HHV8)
gammaherpesvirus with a dsDNA genome
infection of endothelial cells or B cells can lead to malignancy
has a biphasic lifestyle that includes latent infection and lytic replication
both lifecycle phases linked with cancer development
KSHV establishing latency
latent infection: episomal genome, limited gene expression, immune evasion
KSHV lyti replication
lytic reactivation: most viral genes expressed, genome amplification, production of infectious virions
IE genes - transactivating proteins
DE genes - vDNA replication proteins
vDNA replication
late genes - structural proteins
viral episomes -> genome amplification -> infectious virions
activation of the DDR by KSHV
during lytic replication in B cells
ATM activation
AND-PK activation
no Chk1 activation
inhibition of ATR and ATM reduces viral replication efficiency in B cells
localisation of DNA damage sensors/HR repair factors during lytic replication
formation of DNA damage foci during lytic replication
KSHV and DDR
number of DDR proteins are recruited to VRCs including the MRN complex, RPA and Ku80, others like Rad51/Rac52/BRCA1 are localised close to the VRCs but not in them - proteins repairing cellular DNA damaged in some way by KSHV
little evidence for degradation of DDR proteins during KSHV infection
inhibition of Mre11 and ATM reduces the efficiency of viral replication
KSHV appears to inhibit Chk1 phosphorylation
bacteria and the DDR
DNA signalling and repair pathways activated by genotoxic bacteria
many bacteria cause DNA damage in infected cells leading to activation of multiple DDR pathways
bacteria can directly affect DDR pathways e.g. H.pylori reduces expression of p53 MMR and BER proteins
infection with E coli results in DNA damage - partially attributable to the expression of genotoxins encoded by pks island
other toxins e.g. CDT from gram-ve bacteria cause DNA damage
activation of SOS response leads to activation of bacterial DNA repair pathways
Helicobacter pylori and the DDR
common bacteria in the stomach - present in half the population - asymptomatic, causes gastritis in some individuals, chronic infection can lead to stomach cancer
reduced MMR and BER protein expression
reduced p53 due to CagA
CagA also delays prophase and metaphase resulting in incorrect orientation of the spindle and genomic instability
Escherichia coli and the DDR
pathogenicity associated with a pks genomic island - involved in the production of the genotoxin colibactin
activation of the ATM/Chk1/2 and phosphorylation of the Cdc25c leading to cell cycle arrest
DSB occur in some cells infected with pks+ bacteria - usually repaired but in cells deficient in NHEJ can lead to cell death
infection leads to aneuploidy and tetraploidy
other bacteria have pks-like islands and can generate DSBs
cytolethal distending toxin (CDT)
produced by gram-ve bacteria
3 subunits CdtA/CdtB/CdtC
A and C important in internalisation
CDT is secreted into the infected cell prior to binding of the bacteria to the cell surface
causes chromatin fragmentation and DSBs acumulate in cells infected with high doses of these bacteria
nuclear foci form after CDT exposure also activation of ATM-Chk2 and ATR-Chk1 leading to cell cycle arrest and apoptosis
chronic exposure causes genomic instability
it is a radiomimetic agent
SOS response in E. coli
SOS response co-ordinates the response to DNA damage through the LexA repressor
LexA binds the promotor f multiple SOS genes limiting their expression levels
on DNA damage RecA is activated to become a coprotease by the formation of a RecA/ssDNA filament - facilitates self cleavage of LexA
LexA levels decrease then SOS gene expression increases
leads to expression of DNA repair genes
LexA regulates transcription of its own gene as well as the recA gene
increased recA after DNA damage promotes recombination repair and LexA cleavage however increased LexA allows the SOS response to be shut off
