The ability of cells to respond to DNA replication stresses is essential for maintaining genomic integrity and preventing tumorigenesis. Replication stress can be induced by both endogenous and environmental factors and our goal is define the mechanisms by which the mammalian DNA2 nuclease/helicase is able to alleviate different stresses. A multitude of environmental agents that are DNA damaging agents such as aphidicolin, hydroxyurea (HU), camptothecin (CPT), and ionized radiation (IR) can cause replication stress are considered as cancer etiological factors. The DNA replication fork stalls at the damage site and if not stabilized and repaired, the fork will collapse resulting in double stranded DNA breaks that lead to mutations and chromosomal rearrangements, a hallmark of pre-cancerous cells. Replication stresses can also arise due to endogenous factors, such as G rich sequences and highly repeated regions that are difficult to replicate. Especially critical are the telomeres, the specialized DNA-protein structures that protect the chromosome ends from inappropriate degradation and fusion. Mammalian telomeres consist of hundreds to thousands of copies of tandem repeats that terminate in a 3' single-stranded G overhang. The repeats are a source of endogenous replication stress, because the replication fork can stall in the telomeric region. Furthermore, during replication the 3' G overhang is uncapped and resembles a site of DNA damage that can be inappropriately targeted by the DNA damage response machinery. Maintaining telomere integrity is essential; dysfunctional telomeres are generally repaired through chromosomal end-to-end fusions that eventually lead to polyploidy, translocations and cancers. Genetic and molecular studies in yeast indicate that the DNA2 nuclease/helicase, initially thought only to play a role in RNA-DNA flap processing during Okazaki fragment maturation, is involved in stabilizing stalled DNA replication forks, repairing collapsed DNA replication forks and replicating the telomeres. However, when first examined, we found the mammalian DNA2 protein lacked a nuclear localization signal and localized to mitochondria. Yet, our first established DNA2 KO mouse model showed that the most significant phenotype is telomere defects, implicating the entrance of DNA2 into the nucleus. We hypothesize DNA2 is one of the primary nucleases that counteracts DNA replication stresses. Our preliminary data that indicates a specific post-translational modification of the DNA2 protein promotes its localization to the nucleus, where it generates the 3' G overhang of the telomere and protects it from attack by other nucleases and counteracts other replication stresses, and we aim to confirm these mechanisms. In addition, we have preliminary data from mouse models showing that even one missing copy of DNA2 from the genome leads to cancers in high frequency. This is critical information, first because it cannot be inferred from yeast studies, and second, because there are naturally occurring DNA2 SNPs and mutations in human populations and cancer patients. We propose to determine if these mutations predispose mammals to cancer development.

Public Health Relevance

The ability of cells to respond to DNA replication stresses is essential for maintaining genomic integrity and preventing tumorigenesis. DNA replication stresses can be induced by both endogenous factors and environmental insults. Our study aims to define the mechanisms by which mammalian DNA2 nuclease/helicase, a DNA cutting and unwinding protein, is able to alleviate these different types of replication stress to maintain the genome and prevent cancers from developing.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA085344-18
Application #
9182874
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Pelroy, Richard
Project Start
1999-07-01
Project End
2018-11-30
Budget Start
2016-12-01
Budget End
2017-11-30
Support Year
18
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Beckman Research Institute/City of Hope
Department
Type
DUNS #
027176833
City
Duarte
State
CA
Country
United States
Zip Code
91010
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