Distinct Genomic Instabilities Associated with Deficiencies in Homologous Recombination Or the Fanconi Anemia FANCD2 Monoubiquitination Pathway

Distinct genomic instabilities associated with deficiencies in homologous recombination or the Fanconi anemia FANCD2 monoubiquitination pathway

April 19th, 2005

John Hinz Lawrence Livermore National Laboratory Livermore, CA Summary:

Homologous Recombination Fanconi anemia

Genome stability

Model discussion Homologous Recombination repair: Repairing DSBs & restarting replication forks

XRCC3 51 BRCA2 XRCC2 51 51C 51D 51 51 5151B 51 51 Fanconi anemia complementation groups:

FancA, B, C, D1, D2, E, F, G, L

51 BRCA2 51 5151 51 51 51 FA Proteins in the cell

A BA GG Ub C C B FF D2 E L E L A G Ub C B F D2 E L BRCA1 51 BRCA2 51 5151 51 51 51 Construction of a FancG knockout

AA8AA8 FancG gene

KO40KO40 FancGNEO gene

FancGNEO gene BP6BP6 FancG gene BAC Construction of a Rad51D knockout AA8AA8 Rad51D gene Rad51D gene

51D1Lox51D1Lox RadRad51DNEO gene51D gene RadRad51DPURO gene51D gene

51D151D1 Rad gene Rad gene

51D1.351D1.3 Rad51D gene BAC Colony Formation Assays: Cross-link sensitivity

1.0 AA8AA8 KO40KO40 ((fancgfancg)) BP6BP6 Fraction survival

0.1 0.0 1.0 2.0 3.0 4.0 5.0 Mitomycin C (µM) 1.0 AA8AA8 51D1Lox51D1Lox

51D151D1 ((rad51drad51d)) irs1SFirs1SF ((xrcc3xrcc3))

51D1.351D1.3 0.1 KO40KO40 ((fancgfancg)) 0.1 1.0 Mitomycin C (µM) Gene Amplification

Importance of DSBs

Amplifications drive tumorgenesis Amplification Rate at dhfr (units = mutations/dhfr locus/generation)

Cell line Rate (x 10-6) AA8AA8 8.8 ± 0.2 KO40KO40 ((fancgfancg)) 27 ± 2 ( ~3x) BP6BP6 9 ± 2 51D1Lox51D1Lox 7 ± 4 51D151D1 ((rad51drad51d)) 95 ± 4 ( ~10x) irs1SFirs1SF ((xrcc3xrcc3)) 60 ± 14 ( ~7x) 51D1.351D1.3 3 ± 2 Measuring mutation rate at hprt

guanine GMP GTP HPRT 66--thioguaninethioguanine TOXICTOXIC

hprt

Large deletions/rearrangements not detectable FancG mutant has decreased mutation rate

Mutation Rate at hprt (units = mutations/hprt locus/generation)

Cell line Rate (x 10-7) AA8AA8 7 ± 1 KO40KO40 ((fancgfancg)) <2.0 ± .3 ( >3x) BP6BP6 4 ± 1 HR mutants have increased mutation rate

Mutation Rate at hprt Cell line Rate (x 10-7) AA8AA8 77 ±± 11 51D1Lox51D1Lox 5.05.0 ±± .6.6 51D151D1 ((rad51drad51d)) 9090 ±± 2020 (( ~12x)~12x) irs1SFirs1SF ((xrcc3xrcc3)) 220220 ±± 5050 (( ~28x)~28x) 51D1.351D1.3 6.8 ± .8 Hprt mutation spectra

AA8AA8 == ~70%~70% pointpoint mutationsmutations KO40KO40 ((fancgfancg)) == largelarge deletiondeletion (1(1 clone)clone)

51D151D1 ((rad51drad51d)) == allall deletionsdeletions (12(12 clones)clones) Role of FA and HR in mutagenesis?

Model involves replication forks and lesion bypass Replication forks bypassing a lesion: Trans-Lesion Synthesis (TLS)

M TLS

Outcome: possible point mutation Replication forks bypassing a lesion: Fork regression (Chicken foot structure)

Outcome: no mutation Replication forks arresting at a lesion: Fork breakage and collapse

HR NHEJ Model: FA proteins in replication fork stability

TLS M Point HR mutations

HR No mutation NHEJ Few hprt mutants Model: FA proteins in replication fork stability Ub M D2 TLS Point HR mutations

HR No mutation NHEJ Few hprt mutants Model: HR proteins in replication fork stability Ub D2 TLS M Point HR mutations

HR No mutation NHEJ MORE hprt mutants Acknowledgements

Larry Thompson Robert Tebbs Vicki Kopf Eddie Salazar Salustra Urbin Peter Nham