Genome Integrity Discussion Group April 2016

Agenda

* Presentation times are subject to change.

Abstracts

Inner Workings of the UvrA•UvrB DNA Damage Sensor
David Jeruzalmi, PhD, The City College of New York

Efficient elimination of DNA lesions by the nucleotide excision repair (NER) pathway is critical for all organisms. In bacteria, the NER pathway is implemented by the successive action of three proteins, UvrA, UvrB and UvrC via a series of large and dynamic multi-protein complexes. A large number of studies have defined three major stages associated with the early steps of the NER pathway. In stage 1, a large (300–400 kDa) complex of the UvrA and UvrB proteins (AB) scans the genome to identify lesion-containing DNA. This process requires rapid binding and release of DNA; moreover, damage must be specifically recognized, and distinguished from native DNA, despite the fact that the relevant lesions induce widely different DNA structures. Once lesion-containing DNA has been located, it is stably bound by a dimeric form of UvrA within the AB complex (Stage 2). A major reorganization then occurs in which UvrA is lost from the ensemble, and concomitantly, UvrB becomes localized at the site of damage (Stage 3). Following these early stages, additional events lead to excision of the damage on one strand, and repair of the resulting single-stranded gap.
 
We have determined several structures of UvrA and the UvrA•UvrB complex. These structures provoke a critical re-examination of the mechanism of the early stages of bacterial NER, and suggest a revised reaction with several unanticipated features.

A Diffusion Concentration Gradient Model for Replication Origin Firing in Fission Yeast
Atanas Kaykov, The Rockefeller University

We developed a new DNA combing method that allows the investigation of replicating single DNA molecules up to the size of full-length fission yeast chromosomes. Improving the scale of observation by more than one order of magnitude has allowed us to show that the majority of origins fire stochastically forming clusters of closely spaced origins. However, replication origins across the fission yeast genome fire with varying efficiency: some origins are very efficient (fire every other cell cycle) while others are very inefficient (fire every 10–20 cell cycles). We asked if the spatial positioning of replication origins within the nucleus might influence their firing efficiencies.
 
We built a coarse-grained polymer model for fission yeast chromosomes which integrates empirical measurements for: chromosome flexibility, chromosome contact of unlinked loci captured by proximity-ligation techniques (Hi-C), the constrained position of centromeres and mating type locus at the Spindle Pole Body (SPB, centrosome equivalent structure in fungi) and of telomeres at nuclear periphery. The model predicts spatial segregation between efficient and inefficient origins, because efficient origins are located close to the nuclear center while inefficient origins are enriched in the nuclear periphery. This organization is dependent upon histone 3 lysine 9 methylation (H3K9me) since in clr4Δ (histone 3 Lysine 9 methyl transferase) cells the model predicts reciprocal shift in the localization of early and late origins (model integrates Hi-C data established for clr4Δ cells). We tested this prediction of our model by analyzing origin firing efficiencies in clr4Δ cells and showed that origin efficiencies are globally reprogrammed in clr4Δ cells. Specifically, efficient origins became inefficient and some inefficient origins became more efficient. Moreover, our model predicts a correlation between efficiency of origin usage and distance from the SPB. Therefore, we propose a model for stochastic origin firing in which limiting origin activation factors are switched on in the vicinity of SPB and then diffuse throughout the nucleus activating origins of replication.
 
Coauthors: Justin O'Sullivan² and Paul Nurse^1,3.
 
1. Yeast Genetics and Cell Biology, The Rockefeller University.
2. Liggins Institute, University of Auckland.
3. Cell Cycle Laboratory, The Francis Crick Institute, London.

Fan1 Deficiency Results in DNA Interstrand Crosslink Repair Defects, Enhanced Tissue Karyomegaly, and Organ Dysfunction
Supawat Thongthip, The Rockefeller University

Deficiency of FANCD2/FANCI-associated nuclease 1 (FAN1) in humans leads to karyomegalic interstitial nephritis (KIN), a rare hereditary kidney disease, characterized by chronic renal fibrosis, tubular degeneration and characteristic polyploid nuclei in multiple tissues. The mechanism of how FAN1 protects cells is largely unknown but is thought to involve FAN1's function in DNA interstrand crosslink (ICL) repair. Here, we describe a Fan1 deficient mouse and show that FAN1 is required for cellular and organismal resistance to ICLs. We show that the UBZ domain of FAN1, which is needed for interaction with FANCD2, is not required for the initial rapid recruitment of FAN1 to ICL or for its role in DNA ICL resistance. Epistasis analyses reveal that FAN1 has crosslink-repair activities that are independent of the Fanconi anemia proteins and that this activity is redundant with the 5′–3′ exonuclease, SNM1A. Karyomegaly becomes prominent in kidneys and livers of Fan1 deficient mice with age and mice develop liver dysfunction. Treatment of Fan1 deficient mice with ICL-inducing agents results in pronounced thymic and bone marrow hypocellularity and disappearance of c-kit+ cells. Our results provide insight into the mechanism of FAN1 in ICL repair and demonstrate that the Fan1 mouse model effectively recapitulates the pathological features of human FAN1 deficiency.
 
Coauthors: Marina Bellani², Siobhan Gregg¹, Sunandini Sridhar¹, Brooke Conti¹, Yanglu Chen¹, Michael Seidman², and Agata Smogorzewska¹.
 
1. Laboratory of Genome Maintenance, The Rockefeller University.
2. Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health.

Specific and Redundant Roles for Pif1 Helicases in the Maintenance of Genome Integrity
Duncan Smith, PhD, New York University

Pif1 helicases are a conserved family present in both prokaryotes and eukaryotes. While most eukaryotes encode just one, Saccharomyces cerevisiae encodes two distinct Pif1-family helicases—Pif1 and Rrm3—which have been reported to play distinct roles in numerous nuclear processes. We describe the systematic characterization of the roles of Pif1 helicases in replisome progression and lagging-strand synthesis in S. cerevisiae.
 
We demonstrate a redundant role for Pif1 and Rrm3 in stimulating strand-displacement by DNA polymerase δ during lagging-strand synthesis. Further, we show that Rrm3 and to a lesser extent Pif1, facilitate replisome progression past tRNA genes, which in the absence of both helicases become point replication terminators. Although progress past tRNA genes is impeded in the context of both co-oriented and head-on collisions between replication and tRNA transcription, head-on collisions lead to greater replisome stalling: consistent with this observation, we find that tRNA genes in S. cerevisiae are preferentially oriented to minimize head-on replication-transcription conflicts. In contrast to previous reports, we observe no impact of RNA polymerase II-transcribed genes or G-quadruplexes on replisome mobility in pif1 and/or rrm3 mutants.

Travel & Lodging

Our Location

The New York Academy of Sciences

7 World Trade Center
250 Greenwich Street, 40th floor
New York, NY 10007-2157
212.298.8600

Directions to the Academy

Hotels Near 7 World Trade Center

Recommended partner hotel

Club Quarters, World Trade Center
140 Washington Street
New York, NY 10006
Phone: 212.577.1133

The New York Academy of Sciences is a member of the Club Quarters network, which offers significant savings on hotel reservations to member organizations. Located opposite Memorial Plaza on the south side of the World Trade Center, Club Quarters, World Trade Center is just a short walk to the Academy.

Use Club Quarters Reservation Password NYAS to reserve your discounted accommodations online.

Other nearby hotels