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The role of DNA damage tolerance pathways in human cells

Award Number: R01GM134681
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 08/14/2019
PERIOD OF PERFORMANCE END DATE: 03/31/2029

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The role of DNA damage tolerance pathways in human cells - Project Summary Accurate DNA replication is essential for genomic stability and cellular homeostasis, and protects against cellular transformation. Obstacles to DNA replication block the progression of DNA polymerases, arresting replication forks. Unless efficiently restarted, stalled replication forks can collapse, resulting in generation of DNA breaks, promoting genomic instability and carcinogenesis. To avoid fork breakage, cells are equipped with mechanisms to stabilize and restart the fork, thus promoting genomic stability. One important fork restart mechanism involves rapid re-initiation of DNA synthesis downstream of the lesion, through repriming catalyzed by the PRIMPOL enzyme, to restore timely DNA replication. This leaves behind a single stranded (ssDNA) gap in the nascent strand. Accumulation of ssDNA gaps has recently emerged as an important intermediate in DNA damage- induced cell death, thus potentially determining the cellular hypersensitivity to genotoxic agents (including cisplatin), particularly in DNA repair-deficient cells. While by themselves ssDNA gaps are not considered cytotoxic, their accumulation has been associated with formation of cytotoxic double strand DNA breaks (DSBs). DSBs can also cause genomic rearrangements, representing a main driver a chromosomal instability. Understanding the basic mechanisms of ssDNA gap repair is crucial for improving human health. However, how ssDNA gaps are processed into cytotoxic structures represents a major knowledge gap. We have recently uncovered three new processes involved in ssDNA gap homeostasis, which represent the focus of this application. Aim 1 will investigate the regulation of PRIMPOL-mediated fork restart by CAF1. We hypothesize that CAF1 promotes PRIMPOL recruitment to arrested forks to initiate fork repriming. We will measure ssDNA gap formation using specific cell-based assays and test the impact of CAF1 on PRIMPOL recruitment to stressed forks using biochemical and imaging approaches. Aim 2 will investigate the suppression of ssDNA gaps by PARP10. We hypothesize that PARP10, by activating PCNA ubiquitination-mediated TLS, promotes ssDNA gap filling in response to genotoxic exposures. We will test if PARP10 enhances the PCNA ubiquitination-dependent recruitment of TLS polymerases for gap filling using cell-based localization and functional assays, and investigate the functional synergy of PARP10 with the BRCA pathway in chemoresistance. Aim 3 will investigate the nucleolytic processing of ssDNA gaps into cytotoxic structures. We hypothesize that processing of ssDNA gaps into DSBs drives genomic instability and cellular toxicity in certain genetic backgrounds such BRCA deficiency. We will measure the mechanisms and regulation of nuclease engagement to nascent strand gaps in cell-based imaging assays, and how this functionally impacts genome stability and cell death by measuring chromosomal integrity and cellular viability. By investigating three novel ssDNA gap processing mechanisms, using state-of- the-art functional, imaging and genetic approaches, our proposal aims to paint a detailed picture of ssDNA gap homeostasis, with significant implications on our understanding of genome stability and DNA damage sensitivity.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = $615,419 )
2025	2025	PENNSYLVANIA STATE UNIVERSITY	500 UNIVERSITY DRIVE	HERSHEY	PA	17033	DAUPHIN	USA	Biomedical Research and Research Training	000	6	4/22/2025	COMPETING CONTINUATION	$615,419
														Subtotal = $615,419

Issue Date FY: 2024 ( Subtotal = -$1 )
2024	2022	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 EMMET ST N	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	002	5	1/25/2024	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	-$1
2024	2022	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 EMMET ST N	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	001	5	12/3/2023	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$25,613
2024	2022	REGENTS OF THE UNIVERSITY OF MINNESOTA	200 OAK ST SE	MINNEAPOLIS	MN	55455	HENNEPIN	USA	Biomedical Research and Research Training	000	4	12/3/2023	NON-COMPETING CONTINUATION	-$25,613
														Subtotal = -$1

Issue Date FY: 2023 ( Subtotal = -$4,133 )
2023	2022	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 N EMMET ST	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	001	5	1/31/2023	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$145,867
2023	2022	REGENTS OF THE UNIVERSITY OF MINNESOTA	200 OAK ST SE # 224	MINNEAPOLIS	MN	55455	HENNEPIN	USA	Biomedical Research and Research Training	000	4	1/31/2023	NON-COMPETING CONTINUATION	-$150,000
														Subtotal = -$4,133

Issue Date FY: 2022 ( Subtotal = $444,297 )
2022	2022	REGENTS OF THE UNIVERSITY OF MINNESOTA	200 OAK ST SE # 224	MINNEAPOLIS	MN	55455	HENNEPIN	USA	Biomedical Research and Research Training	000	4	5/20/2022	NON-COMPETING CONTINUATION	$444,297
														Subtotal = $444,297

Issue Date FY: 2021 ( Subtotal = $444,297 )
2021	2021	REGENTS OF THE UNIVERSITY OF MINNESOTA	200 OAK ST SE # 224	MINNEAPOLIS	MN	55455	HENNEPIN	USA	Biomedical Research and Research Training	000	3	6/23/2021	NON-COMPETING CONTINUATION	$444,297
														Subtotal = $444,297

Issue Date FY: 2020 ( Subtotal = $444,297 )
2020	2020	REGENTS OF THE UNIVERSITY OF MINNESOTA	200 OAK ST SE # 224	MINNEAPOLIS	MN	55455	HENNEPIN	USA	Biomedical Research and Research Training	000	2	5/22/2020	NON-COMPETING CONTINUATION	$444,297
														Subtotal = $444,297

Issue Date FY: 2019 ( Subtotal = $457,797 )
2019	2019	REGENTS OF THE UNIVERSITY OF MINNESOTA	200 OAK ST SE # 224	MINNEAPOLIS	MN	55455	HENNEPIN	USA	Biomedical Research and Research Training	000	1	8/14/2019	NEW	$457,797
														Subtotal = $457,797

Grand Total All Awards = $2,401,973

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The role of DNA damage tolerance pathways in human cells

Award Number: R01GM134681

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 08/14/2019

PERIOD OF PERFORMANCE END DATE: 03/31/2029

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