SUMMARY DNA damage repair (DDR) pathways modulate cancer risk, progression, and therapeutic response. We systematically analyzed somatic alterations to provide a comprehensive view of DDR deficiency across 33 cancer types. Mutations with accompanying loss of heterozygosity were observed in over 1/3 of DDR genes, including TP53 and BRCA1/2. Other prevalent alterations included epigenetic silencing of the direct repair genes EXO5, MGMT, and ALKBH3 in ~20% of samples. Homologous recombination deficiency (HRD) was present at varying frequency in many cancer types, most notably ovarian cancer. However, in contrast to ovarian cancer, HRD was associated with worse outcomes in several other cancers. Protein structure-based analyses allowed us to predict functional consequences of rare, recurrent DDR mutations. A new machine-learning-based classifier developed from gene expression data allowed us to identify alterations that phenocopy deleterious TP53 mutations. These frequent DDR gene alterations in many human cancers have functional consequences that may determine cancer progression and guide therapy.

SUMMARY Topoisomerase I (TOP1) inhibitors are an important class of anticancer drugs. The cytotoxicity of TOP1 inhibitors can be modulated by replication fork reversal, in a process that requires PARP activity. Whether regressed forks can efficiently restart and the factors required to restart fork progression after fork reversal are still unknown. Here we combined biochemical and electron microscopy approaches with single-molecule DNA fiber analysis, to identify a key role for human RECQ1 helicase in replication fork restart after TOP1 inhibition, not shared by other human RecQ proteins. We show that the poly(ADPribosyl)ation activity of PARP1 stabilizes forks in their regressed state by limiting their restart by RECQ1. These studies provide new mechanistic insights into the roles of RECQ1 and PARP in DNA replication and offer molecular perspectives to potentiate chemotherapeutic regimens based on TOP1 inhibition.

We developed a novel system to create DNA double-strand breaks (DSBs) at defined endogenous sites in the human genome, and used this system to detect protein recruitment and loss at and around these breaks by chromatin immunoprecipitation (ChIP). The detection of human ATM protein at site-specific DSBs required functional NBS1 protein, ATM kinase activity and ATM autophosphorylation on Ser 1981. DSB formation led to the localized disruption of nucleosomes, a process that depended on both functional NBS1 and ATM. These two proteins were also required for efficient recruitment of the repair cofactor XRCC4 to DSBs, and for efficient DSB repair. These results demonstrate the functional importance of ATM kinase activity and phosphorylation in the response to DSBs, and support a model in which ordered chromatin structure changes that occur after DNA breakage depend on functional NBS1 and ATM, and facilitate DNA DSB repair.

Genetic methods of manipulating or eradicating disease vector populations have long been discussed as an attractive alternative to existing control measures because of their potential advantages in terms of effectiveness and species specificity1–3. The development of genetically engineered malaria-resistant mosquitoes has shown, as a proof-of-principle, the possibility of targeting the mosquito’s ability to serve as a disease vector4–7. The translation of these achievements into control measures requires an effective technology to spread a genetic modification from laboratory mosquitoes to field populations8. We have previously suggested that homing endonuclease genes (HEGs), a class of simple selfish genetic elements, could be exploited for this purpose9. Here we demonstrate that a synthetic genetic element, consisting of mosquito regulatory regions10 and the homing endonuclease gene I-SceI11–13, can substantially increase its transmission to the progeny in transgenic mosquitoes of the human malaria vector Anopheles gambiae. We show that the I-SceI element is able to rapidly invade receptive mosquito cage populations, validating mathematical models for the transmission dynamics of HEGs. Molecular analyses confirm that expression of I-SceI in the male germline induces high rates of site-specific chromosomal cleavage and gene conversion, which results in the gain of the I-SceI gene, and underlies the observed genetic drive. These findings demonstrate a new mechanism by which genetic control measures can be implemented. Our results also show in principle how sequence-specific genetic drive elements like HEGs could be used to take the step from the genetic engineering of individuals to the genetic engineering of populations.

The reprogramming of DNA-binding specificity is an important challenge for computational protein design that tests current understanding of protein-DNA recognition, and has considerable practical relevance for biotechnology and medicine [1][2][3][4][5][6] . Here we describe the computational redesign of the cleavage specificity of the intron-encoded homing endonuclease I-MsoI 7 using a physically realistic atomic-level forcefield 8,9 . Using an in silico screen, we identified single basepair substitutions predicted to disrupt binding by the wild-type enzyme, and then optimized the identities and conformations of clusters of amino acids around each of these unfavourable substitutions using Monte Carlo sampling 10 . A redesigned enzyme that was predicted to display altered target site specificity, while maintaining wild-type binding affinity, was experimentally characterized. The redesigned enzyme binds and cleaves the redesigned recognition site ~10,000 times more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original endonuclease. Determination of the structure of the redesigned nuclease-recognition site complex by X-ray crystallography confirms the accuracy of the computationally predicted interface. These results suggest that computational protein design methods can have an important role in the creation of novel highly specific endonucleases for gene therapy and other applications.The nucleotide sequence specificity of DNA-binding proteins can not be deduced directly from amino acid sequence because the packing, hydrogen-bonding and electrostatic interactions responsible for nucleotide-specific recognition are dependent on the threedimensional structure of the protein-DNA complex 11,12 . While a number of canonical amino acid-nucleotide interaction motifs are observed in protein-DNA interfaces 13 , they areCorrespondence and requests for materials should be addressed to J.A. (ashwortj@u.washington.edu) or D.B. (dabaker@u.washington.edu). Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Author Contributions J.J.H. and C.M.D. developed the original protein-DNA interface design methods and code. J.A. made further code and method developments, generated and assessed the computational predictions, and performed mutagenesis, biochemical characterization, and crystallization. D.S. collected and processed the crystallographic data, and aided in protein purification and structure refinement. I-MsoI, which belongs to the LAGLIDADG family of homing endonucleases, is a 170-residue homodimeric enzyme that cleaves long target sites (20-24 base pairs (bp)) with considerable specificity 7,15,16 . The homing endonucleases provide an excellent model system for understanding protein-DNA interaction specificity, as well as starting points for engineering of novel specificities for targeted genomics applications, including gene therapy 4,5 . Crystal structures of the enzymes bound to their recognition sites reveal a rich ...

Werner syndrome (WRN) is an uncommon autosomal recessive disease whose phenotype includes features of premature aging, genetic instability, and an elevated risk of cancer. We used three different experimental strategies to show that WRN cellular phenotypes of limited cell division potential, DNA damage hypersensitivity, and defective homologous recombination (HR) are interrelated. WRN cell survival and the generation of viable mitotic recombinant progeny could be rescued by expressing wild-type WRN protein or by expressing the bacterial resolvase protein RusA. The dependence of WRN cellular phenotypes on RAD51-dependent HR pathways was demonstrated by using a dominant-negative RAD51 protein to suppress mitotic recombination in WRN and control cells: the suppression of RAD51-dependent recombination led to significantly improved survival of WRN cells following DNA damage. These results define a physiological role for the WRN RecQ helicase protein in RAD51-dependent HR and identify a mechanistic link between defective recombination resolution and limited cell division potential, DNA damage hypersensitivity, and genetic instability in human somatic cells.Werner syndrome (WRN) was originally identified among adult siblings in a single family, all of whom displayed cataract formation, premature greying and loss of hair, and scleroderma-like skin changes (48). Further characterization of the clinical, pathological, and genetic aspects of this syndrome following Otto Werner's initial description has led to the recognition of Werner syndrome as an uncommon autosomal recessive disease whose phenotype includes features of premature aging, genetic instability, and an elevated risk of cancer (13,18,39).The WRN gene (also referred to as RECQ3 or RECQL2) was identified by positional cloning in 1996 (51) and was found to encode a 162-kDa member of the human RecQ helicase family with 3Ј35Ј helicase and 3Ј35Ј exonuclease activities. Werner patient mutations truncate the WRN open reading frame and promote loss of the altered protein and both of its associated biochemical activities (4,28,42). Mutations in other human RecQ helicase genes have also been identified in patients with two other genetic instability and tumor predisposition syndromes, Bloom syndrome (12) and Rothmund-Thomson syndrome (24,26).Recently, a homologous recombination (HR) defect in WRN cell lines was identified that included a 25-fold reduction in the rate of generation of viable recombinant daughter cells together with a shift in molecular recombination products from conversion-type to crossover or "popout"-type recombinants that are normally less frequent (35). These analyses focused on spontaneous mitotic recombintion and did not further define the WRN recombination defect or indicate how HR, cell survival, and the response to DNA damage were interrelated in WRN cells. In the work reported here, three different experimental approaches were used to define the WRN recombination defect and the interrelationship of HR and cell survival following DNA damage in WR...

Werner syndrome (WS) is a rare autosomalrecessive disorder characterized by the premature appearance of features of normal aging in young adults. The extensive phenotypic overlap between WS and normal aging suggests they may also share pathogenetic mechanisms. We reported previously that somatic cells from WS patients demonstrate a

The human genome encodes at least 14 DNA-dependent DNA polymerases--a surprisingly large number. These include the more abundant, high-fidelity enzymes that replicate the bulk of genomic DNA, together with eight or more specialized DNA polymerases that have been discovered in the past decade. Although the roles of the newly recognized polymerases are still being defined, one of their crucial functions is to allow synthesis past DNA damage that blocks replication-fork progression. We explore the reasons that might justify the need for so many DNA polymerases, describe their function and mode of regulation, and finally consider links between mutations in DNA polymerases and human disease.