Publications
Wang AT, McHugh PJ. Apollo: a healer of the genome?. Cell Cycle. 2009 Jul 8(13): 1980-1981 [PubMed]
Plummer R, Jones C, Middleton M, Wilson R, Evans J, Olsen AL, Curtin N, Boddy A, McHugh PJ, Newell D, Harris A, Johnson P, Steinfeldt H, Dewji R, Wang D, Robson L, Calvert H. Phase I study of the poly(ADP-ribose) polymerase inhibitor, AG014699, in combination with temozolomide in patients with advanced solid tumors. Clin Cancer Res. 2008 Dec 14(23): 7917-7923 [PubMed]
PURPOSE: One mechanism of tumor resistance to cytotoxic therapy is repair of damaged DNA. Poly(ADP-ribose) polymerase (PARP)-1 is a nuclear enzyme involved in base excision repair, one of the five major repair pathways. PARP inhibitors are emerging as a new class of agents that can potentiate chemotherapy and radiotherapy. The article reports safety, efficacy, pharmacokinetic, and pharmacodynamic results of the first-in-class trial of a PARP inhibitor, AG014699, combined with temozolomide in adults with advanced malignancy. EXPERIMENTAL DESIGN: Initially, patients with solid tumors received escalating doses of AG014699 with 100 mg/m2/d temozolomide x 5 every 28 days to establish the PARP inhibitory dose (PID). Subsequently, AG014699 dose was fixed at PID and temozolomide escalated to maximum tolerated dose or 200 mg/m2 in metastatic melanoma patients whose tumors were biopsied. AG014699 and temozolomide pharmacokinetics, PARP activity, DNA strand single-strand breaks, response, and toxicity were evaluated. RESULTS: Thirty-three patients were enrolled. PARP inhibition was seen at all doses; PID was 12 mg/m2 based on 74% to 97% inhibition of peripheral blood lymphocyte PARP activity. Recommended doses were 12 mg/m2 AG014699 and 200 mg/m2 temozolomide. Mean tumor PARP inhibition at 5 h was 92% (range, 46-97%). No toxicity attributable to AG014699 alone was observed. AG014699 showed linear pharmacokinetics with no interaction with temozolomide. All patients treated at PID showed increases in DNA single-strand breaks and encouraging evidence of activity was seen. CONCLUSIONS: The combination of AG014699 and temozolomide is well tolerated, pharmacodynamic assessments showing proof of principle of the mode of action of this new class of agents.
Hazrati A, Ramis-Castelltort M, Sarkar S, Barber LJ, Schofield CJ, Hartley JA, McHugh PJ. Human SNM1A suppresses the DNA repair defects of yeast pso2 mutants. DNA Repair. 2008 Feb 7(2): 230-238 [PubMed]
Pso2/Snm1 plays a key role in the repair of DNA interstrand cross-links in yeast. Human cells possess three orthologues of Pso2; SNM1A, SNM1B/Apollo and SNM1C/Artemis. Studies using mammalian cells disrupted or depleted for these genes have yielded equivocal evidence that any of these is a true functional homologues of the yeast gene. Here we show that ectopic expression of only one of the three human orthologues, hSNM1A, effectively suppresses the sensitivity of yeast pso2 (snm1) disruptants to cross-linking agents. Two other phenotypes of the pso2 mutants are also partially rescued by ectopic expression of hSNM1A, namely the double-strand repair break defect observed during cross-link processing in pso2 cells, as well as the spontaneous intrachromatid recombination defect of pso2 msh2 double mutants. Finally, we show that recombinant hSNM1A is a 5'-exonuclease, as also recently reported for the yeast Pso2 protein. Together our data suggest that hSnm1A is a functional homologue of yeast Pso2/Snm1.
Kiakos K, Sato A, Asao T, McHugh PJ, Lee M, Hartley JA. DNA sequence selective adenine alkylation, mechanism of adduct repair, and in vivo antitumor activity of the novel achiral seco-amino-cyclopropylbenz[e]indolone analogue of duocarmycin AS-I-145. Mol Cancer Ther. 2007 Oct 6(10): 2708-2718 [PubMed]
AS-I-145 is a novel achiral seco-amino-cyclopropylbenz[e]indolone (seco-amino-CBI) analogue of duocarmycin that has evolved from an alternative strategy of designing CC-1065/duocarmycin agents lacking the characteristic chiral center of the natural agents. The sequence specificity of this compound was assessed by a Taq polymerase stop assay, identifying the sites of covalent modification on plasmid DNA. The adenine-N3 adducts were confirmed at AT-rich sequences using a thermally induced strand cleavage assay. These studies reveal that this compound retains the inherent sequence selectivity of the related natural compounds. The AS-I-145 sensitivity of yeast mutants deficient in excision and post-replication repair (PRR) pathways was assessed. The sensitivity profile suggests that the sequence-specific adenine-N3 adducts are substrates for nucleotide excision repair (NER) but not base excision repair (BER). Single-strand ligation PCR was employed to follow the induction and repair of the lesions at nucleotide resolution in yeast cells. Sequence specificity was preserved in intact cells, and adduct elimination occurred in a transcription-coupled manner and was dependent on a functional NER pathway and Rad18. The involvement of NER as the predominant excision pathway was confirmed in mammalian DNA repair mutant cells. AS-I-145 showed good in vivo antitumor activity in the National Cancer Institute standard hollow fiber assay and was active against the human breast MDA-MD-435 xenograft when administered i.v. or p.o. Its novel structure and in vivo activity renders AS-I-145 a new paradigm in the design of novel achiral analogues of CC-1065 and the duocarmycins.
Lehoczký P, McHugh PJ, Chovanec M. DNA interstrand cross-link repair in Saccharomyces cerevisiae. FEMS Microbiol Rev. 2007 Mar 31(2): 109-133 [PubMed]
DNA interstrand cross-links (ICL) present a formidable challenge to the cellular DNA repair apparatus. For Escherichia coli, a pathway which combines nucleotide excision repair (NER) and homologous recombination repair (HRR) to eliminate ICL has been characterized in detail, both genetically and biochemically. Mechanisms of ICL repair in eukaryotes have proved more difficult to define, primarily as a result of the fact that several pathways appear compete for ICL repair intermediates, and also because these competing activities are regulated in the cell cycle. The budding yeast Saccharomyces cerevisiae has proven a powerful tool for dissecting ICL repair. Important roles for NER, HRR and postreplication/translesion synthesis pathways have all been identified. Here we review, with reference to similarities and differences in higher eukaryotes, what has been discovered to date concerning ICL repair in this simple eukaryote.
McHugh PJ, Sarkar S. DNA interstrand cross-link repair in the cell cycle: a critical role for polymerase zeta in G1 phase. Cell Cycle. 2006 May 5(10): 1044-1047 [PubMed]
DNA interstrand cross-links (ICLs) present a formidable challenge to the cellular repair apparatus, but to date ICL repair pathways have proved difficult to dissect genetically. It now appears that this is partly the result of a high degree of cell cycle phase selectivity in the choice of ICL pathway employed. Here we review recent results showing that Polymerase zeta, specialized translesion plays an important role during ICL repair in G1 phase yeast cells, and that PCNA modification by ubiquitin is a key regulator of its activity. Given that this reaction can occur outside the context of S-phase, these results imply a more general role for PCNA modification in the control of DNA repair pathways through the cell cycle, which is dependent on the type of damage or repair intermediate encountered.
Sarkar S, Davies AA, Ulrich HD, McHugh PJ. DNA interstrand crosslink repair during G1 involves nucleotide excision repair and DNA polymerase zeta. EMBO J. 2006 Mar 25(6): 1285-1294 [PubMed]
The repair mechanisms acting on DNA interstrand crosslinks (ICLs) in eukaryotes are poorly understood. Here, we provide evidence for a pathway of ICL processing that uses components from both nucleotide excision repair (NER) and translesion synthesis (TLS) and predominates during the G1 phase of the yeast cell cycle. Our results suggest that repair is initiated by the NER apparatus and is followed by a thwarted attempt at gap-filling by the replicative Polymerase delta, which likely stalls at the site of the remaining crosslinked oligonucleotide. This in turn leads to ubiquitination of PCNA and recruitment of the damage-tolerant Polymerase zeta that can perform TLS. The ICL repair factor Pso2 acts downstream of the incision step and is not required for Polymerase zeta activation. We show that this combination of NER and TLS is the only pathway of ICL repair available to the cell in G1 phase and is essential for viability in the presence of DNA crosslinks.
Matsushita N, Kitao H, Ishiai M, Nagashima N, Hirano S, Okawa K, Ohta T, Yu DS, McHugh PJ, Hickson ID, Venkitaraman AR, Kurumizaka H, Takata M. A FancD2-monoubiquitin fusion reveals hidden functions of Fanconi anemia core complex in DNA repair. Mol Cell. 2005 Sep 19(6): 841-847 [PubMed]
In DNA damage responses, the Fanconi anemia (FA) protein, FancD2, is targeted to chromatin and forms nuclear foci following its monoubiquitination, a process likely catalyzed by the FA core complex. Here, we show that a chicken FancD2-ubiquitin fusion protein, carrying a Lys-Arg substitution removing the natural monoubiquitination site (D2KR-Ub), could reverse cisplatin hypersensitivity and localize to chromatin in FANCD2-deficient DT40 cells. Importantly, the chromatin targeting was dependent on three core complex components as well as the hydrophobic surface of ubiquitin that may direct protein-protein interactions. Furthermore, a constitutively chromatin bound fusion of D2KR-histone H2B could complement cisplatin sensitivity in FANCD2- but not FANCC-, FANCG-, or FANCL-deficient cells. Thus these core complex components have an additional function in the DNA repair, which is independent of the monoubiquitination and chromatin targeting of FancD2. These results define functional consequences of FancD2 monoubiquitination and reveal previously hidden functions for the FA protein core complex.
Clingen PH, De Silva IU, McHugh PJ, Ghadessy FJ, Tilby MJ, Thurston DE, Hartley JA. The XPF-ERCC1 endonuclease and homologous recombination contribute to the repair of minor groove DNA interstrand crosslinks in mammalian cells produced by the pyrrolo[2,1-c][1,4]benzodiazepine dimer SJG-136. Nucleic Acids Res. 2005 Jun 33(10): 3283-3291 [PubMed]
SJG-136, a pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer, is a highly
efficient interstrand crosslinking agent that reacts with guanine bases
in a 5'-GATC-3' sequence in the DNA minor groove. SJG-136 crosslinks
form rapidly and persist compared to those produced by conventional
crosslinking agents such as nitrogen mustard, melphalan or cisplatin
which bind in the DNA major groove. A panel of Chinese hamster ovary
(CHO) cells with defined defects in specific DNA repair pathways were
exposed to the bi-functional agents SJG-136 and melphalan, and to their
mono-functional analogues mmy-SJG and mono-functional melphalan.
SJG-136 was >100 times more cytotoxic than melphalan, and the
bi-functional agents were much more cytotoxic than their respective
mono-functional analogues. Cellular sensitivity of both SJG-136 and
melphalan was dependent on the XPF-ERCC1 heterodimer, and homologous
recombination repair factors XRCC2 and XRCC3. The relative level of
sensitivity of these repair mutant cell lines to SJG-136 was, however,
significantly less than with major groove crosslinking agents. In
contrast to melphalan, there was no clear correlation between
sensitivity to SJG-136 and crosslink unhooking capacity measured using
a modified comet assay. Furthermore, repair of SJG-136 crosslinks did
not involve the formation of DNA double-strand breaks. SJG-136
cytotoxicity is likely to result from the poor recognition of DNA
damage by repair proteins resulting in the slow repair of both
mono-adducts and more importantly crosslinks in the minor groove.
Barber LJ, Ward TA, Hartley JA, McHugh PJ. DNA interstrand cross-link repair in the Saccharomyces cerevisiae cell cycle: overlapping roles for PSO2 (SNM1) with MutS factors and EXO1 during S phase. Mol. Cell. Biol. 2005 Mar 25(6): 2297-2309 [PubMed]
Pso2/Snm1 is a member of the beta-CASP metallo-beta-lactamase family of
proteins that include the V(D)J recombination factor Artemis.
Saccharomyces cerevisiae pso2 mutants are specifically sensitive to
agents that induce DNA interstrand cross-links (ICLs). Here we
establish a novel overlapping function for PSO2 with MutS mismatch
repair factors and the 5'-3' exonuclease Exo1 in the repair of DNA
ICLs, which is confined to S phase. Our data demonstrate a requirement
for NER and Pso2, or Exo1 and MutS factors, in the processing of ICLs,
and this is required prior to the repair of ICL-induced DNA
double-strand breaks (DSBs) that form during replication. Using a
chromosomally integrated inverted-repeat substrate, we also show that
loss of both pso2 and exo1/msh2 reduces spontaneous homologous
recombination rates. Therefore, PSO2, EXO1, and MSH2 also appear to
have overlapping roles in the processing of some forms of endogenous
DNA damage that occur at an irreversibly collapsed replication fork.
Significantly, our analysis of ICL repair in cells synchronized for
each cell cycle phase has revealed that homologous recombination does
not play a major role in the direct repair of ICLs, even in G2, when a
suitable template is readily available. Rather, we propose that
recombination is primarily involved in the repair of DSBs that arise
from the collapse of replication forks at ICLs. These findings have led
to considerable clarification of the complex genetic relationship
between various ICL repair pathways.
Hartley JA, Spanswick VJ, Brooks N, Clingen PH, McHugh PJ, Hochhauser D, Pedley RB, Kelland LR, Alley MC, Schultz R, Hollingshead MG, Schweikart KM, Tomaszewski JE, Sausville EA, Gregson SJ, Howard PW, Thurston DE. SJG-136 (NSC 694501), a novel rationally designed DNA minor groove interstrand cross-linking agent with potent and broad spectrum antitumor activity: part 1: cellular pharmacology, in vitro and initial in vivo antitumor activity. Cancer Res. 2004 Sep 64(18): 6693-6699 [PubMed]
SJG-136 (NSC 694501) is a rationally designed pyrrolobenzodiazepine
dimer that binds in the minor groove of DNA. It spans 6 bp with a
preference for binding to purine-GATC-pyrimidine sequences. The agent
has potent activity in the National Cancer Institute (NCI) anticancer
drug screen with 50% net growth inhibition conferred by 0.14 to 320
nmol/L (7.4 nmol/L mean). Sensitive cell lines exhibit total growth
inhibition and 50% lethality after treatment with as little as 0.83 and
7.1 nmol/L SJG-136, respectively. COMPARE and molecular target analysis
of SJG-136 data versus that of >60,000 compounds tested in the NCI
60 cell line screen shows that, although the agent has similarity to
other DNA binding agents, the pattern of activity for SJG-136 does not
fit within the clusters of any known agents, suggesting that SJG-136
possesses a distinct mechanism of action. Testing in the NCI standard
hollow fiber assay produced prominent growth inhibition in 20 of 24
i.p. and 7 of 24 s.c. test combinations with 5 of 12 cell lines
exhibiting cell kill. In addition, SJG-136 produced antitumor activity
in mice bearing CH1 and CH1cisR xenografts, a cisplatin-resistant human
ovarian tumor model, and also in mice bearing LS174T xenografts, a
human colon tumor model. SJG-136 produces DNA interstrand cross-links
between two N-2 guanine positions on opposite strands and separated by
2 bp. In human tumor cell lines, the cross-links form rapidly and
persist compared with those produced by conventional cross-linking
agents such as nitrogen mustards. In mice bearing the LS174T human
colon xenograft, DNA interstrand cross-links can be detected in tumor
cells using a modification of the single cell gel electrophoresis
(comet) assay after administration of a therapeutic dose. Cross-links
in the tumor increase with dose and are clearly detectable at 1 hour
after i.v. administration. The level of cross-linking persists over a
24-hour period in this tumor in contrast to cross-links produced by
conventional cross-linking agents observed over the same time period.
Lambert S, Mason SJ, Barber LJ, Hartley JA, Pearce JA, Carr AM, McHugh PJ. Schizosaccharomyces pombe checkpoint response to DNA interstrand cross-links. Mol. Cell. Biol. 2003 Jul 23(13): 4728-4737 [PubMed]
Drugs that produce covalent interstrand cross-links (ICLs) in DNA
remain central to the treatment of cancer, but the cell cycle
checkpoints activated by ICLs have received little attention. We have
used the fission yeast, Schizosaccharomyces pombe, to elucidate the
checkpoint responses to the ICL-inducing anticancer drugs nitrogen
mustard and mitomycin C. First we confirmed that the repair pathways
acting on ICLs in this yeast are similar to those in the main organisms
studied to date (Escherichia coli, budding yeast, and mammalian cells),
principally nucleotide excision repair and homologous recombination. We
also identified and disrupted the S. pombe homologue of the
Saccharomyces cerevisiae SNM1/PSO2 ICL repair gene and found that this
activity is required for normal resistance to cross-linking agents, but
not other forms of DNA damage. Survival and biochemical analysis
indicated a key role for the "checkpoint Rad" family acting through the
chk1-dependent DNA damage checkpoint in the ICL response.
Rhp9-dependent phosphorylation of Chk1 correlates with G(2) arrest
following ICL induction. In cells able to bypass the G(2) block, a
second-cycle (S-phase) arrest was observed. Only a transient activation
of the Cds1 DNA replication checkpoint factor occurs following ICL
formation in wild-type cells, but this is increased and persists in
G(2) arrest-deficient mutants. This likely reflects the fraction of
cells escaping the G(2) damage checkpoint and arresting in the
subsequent S phase due to ICL replication blocks. Disruption of cds1
confers increased resistance to ICLs, suggesting that this second-cycle
S-phase arrest might be a lethal event.
Kiakos K, Howard TT, Lee M, Hartley JA, McHugh PJ. Saccharomyces cerevisiae RAD5 influences the excision repair of DNA minor groove adducts. J Biol. Chem. 2002 Nov 277(46): 44576-44581 [PubMed]
Nucleotide excision repair (NER) is the primary pathway for the removal
of DNA adducts that distort the double helix. In the yeast
Saccharomyces cerevisiae the RAD6 epistasis group defines a more poorly
characterized set of DNA damage response pathways, believed to be
distinct from NER. Here we show that the elimination of the DNA minor
groove adducts formed by an important class of anticancer antibiotic
(CC-1065 family) requires NER factors in S. cerevisiae. We also
demonstrate that the elimination of this class of minor groove adduct
from the active MFA2 gene depends upon functional Rad18 and Rad6. This
is most clear for the repair of adducts on the transcribed strand,
where an absolute requirement for Rad6 and Rad18 was seen. Further
experiments revealed that a specific RAD6-RAD18-controlled subpathway,
the RAD5 branch, mediates these events. Cells disrupted for rad5 are
highly sensitive to this minor groove binding agent, and rad5 cells
exhibit an in vivo adduct elimination defect indistinguishable from
that seen in rad6 and rad18 cells as well as in NER-defective cells.
Our results indicate that the RAD5 subpathway may interact with NER
factors during the repair of certain DNA adducts.
