Genome Integrity Discussion Group June 2016

Agenda

* Presentation times are subject to change.

Abstracts

Reconstituting Chromosome Replication
John Diffley, Francis Crick Institute, Clare Hall Laboratory, South Mimms, United Kingdom

The eukaryotic cell cycle coordinates the accurate duplication and segregation of the genome during proliferation. The large genomes of eukaryotic cells are replicated from multiple replication origins during S phase. These origins are not activated synchronously at the beginning of S phase, but instead fire throughout S phase according to a pre-determined, cell type specific program.
 
Ensuring that each origin is efficiently activated once and only once during each S phase is crucial for maintaining the integrity of the genome. This is achieved by a two-step mechanism. The first step, known as licensing, involves the loading of the Mcm2-7 proteins into pre-replicative complexes (pre-RCs) at origins. In the second step, the MCM helicase is activated by a large set of 'firing factors'. These two steps are differentially regulated by cyclin dependent kinase (CDK): licensing is inhibited by CDK, whilst firing requires CDK. As a consequence, licensing can only happen during G1 phase, when CDK activity is low, and origin firing cannot occur during G1 phase.
 
We have recently described the reconstitution of the initiation of eukaryotic DNA replication with purified proteins. I will describe recent results on the reconstitution the entire replisome, and will describe how chromatinised templates are replicated in vitro.

Dissecting Substrate Dependent Mechanisms of Cdc7-mediated Replication Fork Protection Using Chemical Genetics and Quantitative Phosphoproteomics
Mathew J. Jones, Molecular Biology Program, Sloan Kettering Institute

The uncontrolled proliferation of cancer cells places a large amount of stress on the cellular machinery required for DNA replication. In response, cancer cells frequently overexpress replication components such as the Dbf4-dependent kinase, Cdc7. Small molecule Cdc7 inhibitors have shown encouraging results in preclinical tumor models; however, their broad specificity and weak penetrance in vivo prohibit the functional assessment of Cdc7. Cdc7 plays a vital role in initiating DNA replication, via the phosphorylation and activation of the MCM replicative helicase. In contrast, little is known about Cdc7's later functions and substrates during DNA synthesis. To address these issues, we employed adeno-associated virus (AAV) vectors to delete and replace the Cdc7 gene (CDC7L1) in human somatic cells with a "shokat" allele that can be rapidly and specifically inhibited by small molecule bulky purine analogs. Using this approach, we have discovered that Cdc7 is required to preserve the stability and restart potential of stalled replication forks. To understand the mechanisms underlying this regulation, we compared "Cdc7-on" and "Cdc7-off" cells using SILAC and quantitative phosphoproteomics. We identified novel Cdc7-dependent phosphorylation sites on the cohesin ring complex and a component of the BRCA1-A complex. Using CRISPR-mediated gene-editing to generate stable knockout cell lines for these substrates, we found that the Cdc7-phosphorylated forms of these substrates make complementary contributions to fork protection: whereas cohesin stabilizes nascent strands during the stall, the BRCA1-A complex promotes restart after the block to elongation is removed. Our findings demonstrate a novel role for Cdc7 after initiation, at stalled replication forks. Since replication fork stalling is elevated upon oncogenic transformation and chemotherapy treatments, these insights may be relevant to exploiting and optimizing Cdc7 inhibition therapeutically.
 
Coauthors: Stephanie Munk², Amnon Koren³, Steve McCarroll⁴, Jesper Olsen², and Prasad V. Jallepalli¹.
 
1. Sloan Kettering Institute, Molecular Biology Program.
2. University of Copenhagen and Novo Nordisk Foundation, Center for Protein Research.
3. Cornell University, Department of Molecular Biology and Genetics.
4. Harvard Medical School, Department of Genetics.

Opposing Recombination Position Effects Regulate Copy Number of the Tandem Repetitive rDNA Array
Andrés Mansisidor, Hochwagen Lab, New York University

Repetitive DNA arrays are prone to non-allelic homologous recombination (NAHR), which greatly influences their stability. NAHR dynamics within DNA arrays, however, remain elusive due to the difficulty of distinguishing individual repeats. To address this experimental limitation, we utilized a collection of repeat-specific insertion tags that are tiled throughout the ribosomal DNA (rDNA) array of Saccharomyces cerevisiae. We discovered that NAHR is not only subject to different levels, but also distinct modes of recombination based on repeat position within the rDNA. Intrachromosomal recombination is enriched at border repeats and is coupled to a stable array size. In contrast, condensin mutant ycs4-2 induces an opposing recombination position effect that increases a distinct mode of NAHR specifically at central repeats that is coupled to array expansion. This finding of position-dependent NAHR brings to light an unexpected architectural feature of the highly conserved rDNA array that likely provides a key regulatory link between global cellular metabolism and genome integrity.

The Genomic Repercussions of RAD5 Overexpression
Robert J.D. Reid, Department of Genetics & Development and Department of Systems Biology, Columbia University Medical Center

The human HLTF gene is located on chromosome 3q24 and is frequently amplified in squamous cell lung carcinomas. HLTF is the human ortholog of the Saccharomyces cerevisiae RAD5 gene, a DNA helicase and ubiquitin ligase that functions in post replication DNA damage repair. Overexpression of RAD5 in yeast results in slow growth and genome instability. Additionally, RAD5 overexpression sensitizes cells to DNA damaging agents that slow replication forks. To better understand the consequences of RAD5 overexpression, we used Selective Ploidy Ablation (SPA), a rapid high throughput plasmid screening protocol, to identify Saccharomyces cerevisiae genes whose function becomes essential when RAD5 is overexpressed. Many DNA replication and DNA double strand break repair genes were identified covering multiple steps in these processes, including replication fork protection, lagging strand DNA synthesis, DNA end resection, homology dependent strand invasion and resolution of Holliday junction intermediates. Curiously, genes regulating the process of post replication repair (PRR) are unaffected in the screen. Cells encountering DNA damage that results in replication fork stalling enter the PRR pathways via modification of PCNA by ubiquitylation. Using multiple mutant backgrounds, we confirmed that ubiquitylation of PCNA is not necessary for the slow growth phenotype resulting from RAD5 overexpression. Since Rad5 is a multidomain polypeptide containing helicase, ubiquitin ligase and DNA binding functions, we investigated the contribution of each of these domains to RAD5 toxicity. Each domain was mutated and then overexpressed in an array of strains deficient for specific repair functions. Automated quantitative analysis of the growth curves was used to define the effects of each RAD5 domain mutant. We find that the individual domains of overexpressed RAD5 have separate contributions to cell toxicity in different genetic backgrounds. As the function of Rad5 is conserved in human cells, the identified SDL interactions may help to develop targeted therapeutic approaches for squamous cell carcinomas of the lung.
 
Coauthors: Eric Bryant, Ivana Sunjevaric, and Rodney Rothstein, Department of Genetics & Development and Department of Systems Biology, Columbia University Medical Center.

New Insights into Homologous Recombination Using Single Molecule Super Resolution Imaging
Donna R. Whelan, Department of Biochemistry and Molecular Pharmacology, New York University Medical Center

Collapsed replication forks resulting in one-sided double strand breaks (DSBs) are one of the most dangerous types of genomic stress and have previously been demonstrated as the predominant endogenous target of homologous recombination (HR)¹. Despite the clear importance of the HR pathway in maintaining genomic integrity with relation to oncogenesis, immune diseases, and in cancer treatment, elucidation of the over-arching physical organization and progression of HR machinery has proven elusive². This is in part due to a previous inability to directly visualize these events in situ because of the diffraction limit of light. Here we have applied novel super resolution (SR) assays for imaging of single replication forks under stress conditions in order to detect the generation and HR repair of DSBs. These endogenous-like one-sided DSBs are induced using camptothecin to trap the Topoisomerase I cleavage complex resulting in DSBs similar to those that occur endogenously during S phase¹. After damage, we monitored the repair of these DSBs over 16 hours in order to uncover both kinetic and structural information regarding the HR process. In particular we have tracked the arrival and interactions of several principal repair proteins at DSBs—Ku, MRE11, CtIP, BRCA1, BRCA2, RAD51, RPA and BLM—as well as directly detected DSBs using the TUNEL assay, nascent DNA using Click chemistry, and single stranded DNA using undenatured genome-incorporated BrdU. The generated SR images were analyzed using in-house protocols for unbiased pair correlation, particle overlap with Monte Carlo random simulations, and individual and inter-foci colocalization analyses. Most interestingly, in light of current debate, we definitively show the loading of both Ku and MRE11 onto the same DSB; the variety and interaction of MRE11, BRCA1, and CtIP complexes and monomers involved in processing and resection; the simultaneous occupation of DSB DNA several hours after damage by MRE11, RPA and RAD51, and later by RAD51 and RPA in a dynamic process that persists until repair is complete²⁻³. Together these observations provide new insights into HR repair, while also establishing innovative SR assays that will allow for expansive future refinement of DSB repair models.
 
1. Saleh-Gohari, et al. (2005) Molecular and Cellular Biology 25, 7158-7169.
2. Renkawitz, J., Lademann, C. A., and Jentsch, S. (2014) Nature Reviews Molecular Cell Biology 15, 369-383.
3. Bunting, S. F., and Nussenzweig, A. (2013) Nature Reviews Cancer 13, 443-454.
 
Coauthors: Yandong Yin, Keria Bermudez-Hernandez, Sarah Keegan, David Fenyo, and Eli Rothenberg, Department of Biochemistry and Molecular Pharmacology, New York University Medical Center.

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