Tuesday, July 1, 2025 7/1/2025

Mechanisms for the high fidelity of translesion synthesis by Y-family DNA polymerases in human cells

Award Number: R35GM148364
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: 02/01/2023
PERIOD OF PERFORMANCE END DATE: 11/30/2027

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Mechanisms for the high fidelity of translesion synthesis by Y-family DNA polymerases in human cells - ABSTRACT By promoting replication through DNA lesions, translesion synthesis (TLS) DNA polymerases (Pols) play a critical role in preventing chromosomal instability and protecting against tumorigenesis. Unlike replicative Pols, TLS Pols have less constrained active sties and they lack proofreading 3'→5' exonuclease activity. Consequently, purified TLS Pols synthesize DNA opposite DNA lesions with an extremely low fidelity. Despite this, TLS operates in a predominantly error-free manner in normal human cells (not derived from cancers), The overall objective in this project is to identify the cellular processes and mechanisms by which high fidelity is imposed upon TLS by the intrinsically highly error-prone Y-family Pols. Using a combination of genetic, cellular, biochemical, and structural approaches, we will address the following questions: (1) Do the Y-family Pols associate with other protein factors in a multiprotein ensemble and do these proteins have activities that elevate the fidelity of the TLS Pol? (2) What is the protein composition of the entire Y-family Pol ensemble for error-free TLS in human cells? (3) How is the fidelity of TLS modulated by the components of the multiprotein ensemble? (4) What are the molecular underpinnings of action mechanisms via which components of the multiprotein ensemble impose high fidelity on Y-family Pols? To pursue these questions, we have identified a number of protein factors that function in TLS specifically in conjunction with Y-family Pols; included among these proteins are WRN which possesses DNA helicase and 3'→5' exonuclease activities, and WRNIP1 which has a DNA dependent ATPase activity. How these activities contribute to the fidelity of TLS by Y-family Pols opposite different types of DNA lesions will be analyzed in extensive mutational studies that include genome wide sequencing. Using proximity labeling in which TurboID is fused to Polη, we will determine whether there are additional proteins that function in TLS in conjunction with Y-family Pols and whether activities in these proteins affect the fidelity of TLS by these Pols. In biochemical studies with the purified multiprotein ensemble of Polη or Polι, we will ascertain the roles of WRN 3'→5' exonuclease, WRN and WRNIP1 ATPase, and of any other newly identified activities in the high fidelity of TLS by these Pols opposite different types of DNA lesions. From cryo- EM studies with the purified multiprotein ensemble of Polη or Polι, we will determine mechanistically how the components of the multiprotein ensemble modulate the fidelity of these Y-family Pols opposite DNA lesions. Cumulatively, these studies will identify the components of the multiprotein Y-family TLS replicases which carry out high fidelity TLS in human cells. They will reveal the mechanisms by which the various components constrain Y-family Pols' active sites to restrain nucleotide (nt) misincorporation and how WRN's 3'→5' exonuclease activity is coordinated with the TLS Pol for the removal of misinserted nt. These studies will be paradigm shifting and will open new vistas of research into the mechanistic details of TLS Pols' fidelity and they will give impetus to further elaboration of the roles of TLS Pols in genome integrity.


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 = $388,626 )
2025	2025	UNIVERSITY OF TEXAS MEDICAL BRANCH AT GALVESTON	301 UNIVERSITY BLVD	GALVESTON	TX	77555	GALVESTON	USA	Biomedical Research and Research Training	001	3	11/21/2024	NON-COMPETING CONTINUATION	$388,626
2025	2024	UNIVERSITY OF TEXAS MEDICAL BRANCH AT GALVESTON	301 UNIVERSITY BLVD	GALVESTON	TX	77555	GALVESTON	USA	Biomedical Research and Research Training	000	2	11/15/2024	NON-COMPETING CONTINUATION	$0
														Subtotal = $388,626

Issue Date FY: 2024 ( Subtotal = $431,806 )
2024	2024	UNIVERSITY OF TEXAS MEDICAL BRANCH AT GALVESTON	301 UNIVERSITY BLVD	GALVESTON	TX	77555	GALVESTON	USA	Biomedical Research and Research Training	001	2	6/21/2024	NON-COMPETING CONTINUATION	$43,180
2024	2024	UNIVERSITY OF TEXAS MEDICAL BRANCH AT GALVESTON	301 UNIVERSITY BLVD	GALVESTON	TX	77555	GALVESTON	USA	Biomedical Research and Research Training	000	2	11/9/2023	NON-COMPETING CONTINUATION	$388,626
														Subtotal = $431,806

Issue Date FY: 2023 ( Subtotal = $431,806 )
2023	2023	UNIVERSITY OF TEXAS MEDICAL BRANCH AT GALVESTON	301 UNIVERSITY BLVD	GALVESTON	TX	77555	GALVESTON	USA	Biomedical Research and Research Training	000	1	1/17/2023	NEW	$431,806
														Subtotal = $431,806

Grand Total All Awards = $1,252,238

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Mechanisms for the high fidelity of translesion synthesis by Y-family DNA polymerases in human cells

Award Number: R35GM148364

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: 02/01/2023

PERIOD OF PERFORMANCE END DATE: 11/30/2027

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