3rd Microbial Genome Maintenance, OSLO, August30-Sept.2, 2008

NER dependent MutagenesisRobert P FUCHS, CNRS Marseille

m

Replication fork context

TLS pathways in E. coli: switching among polymerases

Replicative Polymerase

Specialized Polymerases

“TLS patch”Pol III

IVV

Pol III

VPol III

II

4. Depending on the lesion and/or its sequence context one or a combination oftwo specialized DNA pol’s may be needed.

2. Polymerase recruitment to the beta-clamp is required for TLS in vivo.

3. Recruitment is a “random” process (mass action law).

1. E. coli has 3 SOS Pol’s: Pol II (polB), Pol IV (dinB) and Pol V (umuDC) All 3 are involved in TLS; Pol V being the major TLS polymerase

Lesion Sequence context Error-free TLS Mutagenic TLS

T(6-4)T 5’-AATT- Pol V Pol V (T->C)

BaP 5’-GGG*- Pol V + Pol IV Pol V + Pol IV (-1)

AAF 5’-GGG*- Pol V Pol V (-1)

AAF 5’-gGCG*Cc Pol V Pol II (-2)

AAF 5’-gGCGCGCc ? Pol II or Pol V (-2)Napolitano et.al., (2000) EMBO J. 19, 6259-65 ; Wagner et.al., DNA Repair 1(2002)159-67.

TLS pathways : ‘lesion / sequence context’ effects

rifR mutation assay :1. Target gene: rpoB gene (the β su of RNA polymerase)

2. Wt RNA polymerase is inhibited by rifampicin (ie rifampicin kills wt cells)a rifR mutant is no longer inhibited by rif but still encodes a functional RNApol.

3. 95% of rifR mutants map within aa 500-580 (ie a 240 nt long DNA sequence)

4. RifR mutants: 46 aa substitutions distributed over 27 distinct base positions representing 18 distinct aa have been identified in 900 rifR mutants. All six bs are found (transversions/transitions 40/60) (Krokan, 2000).

Exponentially growing culture.

UVExpression time (0-6h) Plate on LB

Plate of rif ()Mf = rifR/survivors.

Is there a specific molecular signature for each of the two sub-pathways ?

Pol VPol IV

m

m

UV-induced mutation spectrum in wild-type strains

Transition G.C -> A.T A.T -> G.CTransversion A.T -> T.A G.C -> T.A G.C -> C.G A.T -> C.G

526 531 533

GCA GTG AAA GAG TTC TTC GGT TCC AGC CAG CTG TCT CAG TTT ATG GAC CAG AAC AAC CCG CTG TCT GAG ATT ACG CGT CAC TTT CTC AAG AAG CCA AGG TCG GTC GAC AGA GTCAAA TAC CTG GTC TTG TTG GGC GAC AGA CTC TAA TGC

CAC AAA CGT CGT ATC TCC GCA CTC GGC CCA GGC GGT CTG ACC CGT GAA CGT GCA GGC TTC GAA GTT CGA GAC GTA GTG TTT GCA GCA TAG AGG CGT GAG CCG GGT CCG CCA GAC TGG GCA CTT GCA CGT CCG AAG CTT CAA GCT CTG CAT

CAC CCG ACT CAC TAC GGT CGC GTA TGT CCA ATC GAA ACC CCT GAA GGT CCG AAC ATC GGT CTG ATC AAC TCT CTGGTG GGC TGA GTG ATG CCA GCG CAT ACA GGT TAG CTT TGG GGA CTT CCA GGC TTG TAG CCA GAC TAG TTG AGA GAC

512 513 516 525

564 572

5’Py-Py(6-4)3’

5’Py^Py(CPD)3’

3’

5’

3’

5’

UV-induced mutation spectrum in ΔpolB ΔdinB strains

GCA GTG AAA GAG TTC TTC GGT TCC AGC CAG CTG TCT CAG TTT ATG GAC CAG AAC AAC CCG CTG TCT GAG ATT ACG CGT CAC TTT CTC AAG AAG CCA AGG TCG GTC GAC AGA GTC AAA TAC CTG GTC TTG TTG GGC GAC AGA CTC TAA TGC

CAC AAA CGT CGT ATC TCC GCA CTC GGC CCA GGC GGT CTG ACC CGT GAA CGT GCA GGC TTC GAA GTT CGA GAC GTA GTG TTT GCA GCA TAG AGG CGT GAG CCG GGT CCG CCA GAC TGG GCA CTT GCA CGT CCG AAG CTT CAA GCT CTG CAT

CAC CCG ACT CAC TAC GGT CGC GTA TGT CCA ATC GAA ACC CCT GAA GGT CCG AAC ATC GGT CTG ATC AAC TCT CTGGTG GGC TGA GTG ATG CCA GCG CAT ACA GGT TAG CTT TGG GGA CTT CCA GGC TTG TAG CCA GAC TAG TTG AGA GAC

Transition G.C -> A.T A.T -> G.CTransversion A.T -> T.A G.C -> T.A G.C -> C.G A.T -> C.G

512 513 516

531 533

564 574

Leading strand / Transcribed strand

Lagging strand / Non-Transcribed strand

5’Py-Py(6-4)3 ’

 5’Py^Py(CPD)3 ’

5’

3’

3’

5’

Fig. 6c

DNA sequence specificity in wild-type and ΔpolB ΔdinB strains

Mutation type Spontaneous mutations

UV-induced mutations in a wild-type strain

UV-induced mutations in a ΔpolB ΔdinB strain

Transition 479 (63%) 29 (62%) (100%) 34 (72%) (100%)G.C -> A.T 244 (32%) 24 (51%) (83%) 32 (68%) (94%)A.T -> G.C 235 (31%) 5 (11%) (17%) 5 (4%) (6%)

Transversion 284 (37%) 18 (38%) (100%) 13 (28%) (100%) A.T -> T.A 71 (9%) 9 (19%) (50%) 4 (9%) (30%)G.C -> T.A 142 (19%) 6 (13%) (33%) 7 (15%) (54%)G.C -> C.G 11 (1%) 2 (4%) (11%) 1 (2%) (8%)A.T -> C.G 60 (8%) 1 (2%) (6%) 1 (2%) (8%)

Single mutations 763 (100%) 47 (85%) 47 (87%)Double mutations 6 (11%) 3 (6%)Triple mutations 1 (2%) 0no mutation found in 1 (2%) 4 (7%)the sequenced regionTotal of mutants 763 (100%) 55 (100%) 54 (100%) sequenced

Fig. 6a

Both wild type and polBdinB strains exhibit a similar mutagenicsignature (typical for Pol V and UV-light).

Q: Is DNA repair involved in the Pol IV dependent pathway ?

Pol VPol IV

m

mReplication dependent pathway

UV-induced mutation spectrum in wt versus polBdinB

Pol V m

m

Pol IV (Pol II)

UvrA, UvrC, Cho

Replication pathway (R-IM)

NER pathway (NER-IM)

….mutations to strep resistance occurred in the absence of DNAreplication in a uvr+ but not in a uvrA strain….

…. following UV, the majority of forward mutations induced in RAD+ occur before the first cell division, whereas in rad1-1 the mutationsonly appear after the cell has divided and DNA replication has occurred….

Pol V m

m

Pol IV (Pol II)

UvrA, UvrC, Cho

Q: in the NER pathway is there a requirement for the β-clamp ?

Replication pathway (R-IM)

NER pathway (NER-IM)

Pol IV (dinB) requires interaction with the β-clampfor TLS in vivo

---345GQLVLGL351

Pol IV (wild type)---356GQ346LVLGLPol IV Δ5 (β- mutant)

Becherel et al, DNA Repair (2002)

Assay: TLS of aG-BaP adductin vivo

seen on density maps

The C-terminal peptide (P16) of Pol IV binds to the β-clamp (structure resolved at 1.65 A)

P16: VTLLDPQMERQLVLGL

Burnouf et al, JMB 2004

Pol V m

m

Pol IV (Pol II)

UvrA, UvrC

Replication pathway (R-IM)

NER pathway (NER-IM)

β-clamp

Q: can we see a different kinetics of induction ?

β-clamp

Pol V m

m

Pol IV (Pol II) β-clamp

UvrA, UvrC

Replication pathway (R-IM)

NER pathway (NER-IM)

β-clamp

Q: does mutagenesis occur in the absence of DNA replication ?

dnaB8ts : a replication quick stop mutant strain

DnaGDnaB

DnaB hexamer unwinds the parental strands at the replication forkDnaB recruits the DnaG primaseDnaB interacts with the tau sub-unit of Pol III HE

dnaB8ts : normal replication at 30-35°C; at 42°C: no replication. dnaB8ts is a point mutation ala130val: ATPase and helicase deficient

5’

5’

NER5’

5’

5’Gap enlargement (via 3’-> 5’ exo: xonA, exoX, xseA..)

5’

5’

5’m

5’

5’

Gap filling

Pol IV (or Pol II) for gap-filling Pol V (TLS step)

NER-Induced Mutagenesis (NER-IM): a model involving a secondary lesion located in the excision gap

Q: what is the dose-response curve for the NER pathway ?

two-hit model ?

mf= f(dose2)

y = 1,4993x - 7880R2 = 0,9781

0

5000

10000

15000

20000

25000

30000

35000

0 5000 10000 15000 20000 25000 30000

(dose J/m2)2

mu

t fr

eq

uen

cy

wt (moyenne)

Linéaire (wt (moyenne))

Conclusion: in the wild type strain the response is quadratic: two-hit model

Conclusions / remaining questions:

NER-IM defines a new replication-independent mutation pathway(significance for resting cells ?)

How are gaps enlarged ? Accidentally or in a controlled manner ?

A new function for Pol IV in filling these large gaps ? Why is Pol IV (Pol II) specifically required ?

Genome Instability and Carcinogenesis CNRS, Marseille, FranceAgnès TissierShingo FujiiAsako ISOGAWAStephane CoulonGerard MazonGaelle PhilippinStephanie GonCarole Alies

Regine JANELRita NAPOLITANO