The long-term goal of our laboratory is to elucidate the molecular mechanisms of DNA replication, repair and recombination by studying the DNA joining step that is common to these different DNA transactions. There are three genes encoding DNA ligases in human cells. The participation of these enzymes in different cellular functions is directed by specific protein-protein interactions with different partner proteins. In this proposal we are focused on human DNA ligase I (hLigI), which plays a key role in DNA replication and DNA repair. Notably, both deficiency and overexpression of hLigI cause genomic instability.
In Specific Aim 1, we will focus on defining at the molecular level the abnormalities at the replication fork that are caused by hLigI deficiency. These studies will provide novel insights into the relationship between the joining of Okazaki fragments, nucleosome assembly and chromatin maturation. Interestingly, reduced hLigI activity and reduced activity of the Elg1-RFC clamp loader, which is involved in the maintenance of genomic stability, both result in increased association of PCNA with DNA. In addition, we have discovered an interaction between hLigI and the Elg1- RFC complex in preliminary studies.
In Specific Aim 2, we will test the linked hypotheses that the physical and functional interplay between hLigI and the Elg1-RFC complex links the ligation of Okazaki fragments with PCNA unloading and that defects in PCNA unloading cause abnormalities in nucleosome assembly and chromatin maturation. The observations that elevated levels of hLigI cause genomic instability provides a compelling rationale for delineating the mechanisms that regulate the cellular levels of hLigI. In preliminary studies, we have identified DCAF7, a substrate specificity factor of the Cullin-DDB1 ubiquitin ligase, as a hLig1 interacting protein and show that hLigI is degraded by the ubiquitin-proteasome pathway in response to both DNA damage and inhibition of cell proliferation. The role of hLigI ubiquitylation by the Cullin-DDB1 ubiquitin ligase in regulating the steady state levels of hLigI in response to both DNA damage and inhibition of cell proliferation will be explored in Specific Aim 3. The proposed studies will provide novel insights into the mechanisms and regulation of pathways that play a critical role in maintaining genome stability and preventing cancer formation. In addition, this information will provide the framework for characterizing abnormalities in hLigI-dependent genome maintenance pathways and determining how they contribute to malignant phenotype of tumor cells.

Public Health Relevance

It is well established that genomic instability drives the progression of a normal cell into a cancer cell. Human cells have a complex network of pathways that act together to maintain genome stability. This study is focused on an enzyme, DNA ligase I, that joins breaks in DNA that occur normally during DNA replication and as a consequence of DNA damage. Notably, mice deficient in DNA ligase I have increased genome instability and an increased incidence of cancer, indicating that this enzyme plays a critical role in preventing cancer formation. Furthermore, it has been shown that overexpression of DNA ligase I also causes genomic instability and that overexpression of DNA ligase I occurs frequently in cancers, highlighting the importance of understanding the mechanisms that regulate the cellular levels of DNA ligase I. The proposed studies will provide fundamental insights into the mechanisms and regulation of DNA replication and the abnormalities that underlie tumor formation in both DNA ligase I deficiency and overexpression. The differences between normal and cancer cells in the mechanisms that maintain genomic stability offer an opportunity to develop therapeutic strategies that selectively target cancer cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM057479-15A1
Application #
8695937
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Barski, Oleg
Project Start
2000-01-01
Project End
2018-07-31
Budget Start
2014-08-15
Budget End
2015-07-31
Support Year
15
Fiscal Year
2014
Total Cost
$280,000
Indirect Cost
$75,534
Name
University of New Mexico Health Sciences Center
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
829868723
City
Albuquerque
State
NM
Country
United States
Zip Code
87131
Slean, Meghan M; Panigrahi, Gagan B; Castel, Arturo López et al. (2016) Absence of MutSβ leads to the formation of slipped-DNA for CTG/CAG contractions at primate replication forks. DNA Repair (Amst) 42:107-18
Peng, Zhimin; Liao, Zhongping; Matsumoto, Yoshihiro et al. (2016) Human DNA Ligase I Interacts with and Is Targeted for Degradation by the DCAF7 Specificity Factor of the Cul4-DDB1 Ubiquitin Ligase Complex. J Biol Chem 291:21893-21902
Greco, George E; Matsumoto, Yoshihiro; Brooks, Rhys C et al. (2016) SCR7 is neither a selective nor a potent inhibitor of human DNA ligase IV. DNA Repair (Amst) 43:18-23
Hegde, Pavana M; Dutta, Arijit; Sengupta, Shiladitya et al. (2015) The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome: DOMINANT NEGATIVE FUNCTION OF THE CTD. J Biol Chem 290:20919-33
Hegde, Muralidhar L; Hegde, Pavana M; Bellot, Larry J et al. (2013) Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins. Proc Natl Acad Sci U S A 110:E3090-9
Tomkinson, Alan E; Howes, Timothy R L; Wiest, Nathaniel E (2013) DNA ligases as therapeutic targets. Transl Cancer Res 2:
Peng, Zhimin; Liao, Zhongping; Dziegielewska, Barbara et al. (2012) Phosphorylation of serine 51 regulates the interaction of human DNA ligase I with replication factor C and its participation in DNA replication and repair. J Biol Chem 287:36711-9
Howes, Timothy R L; Tomkinson, Alan E (2012) DNA ligase I, the replicative DNA ligase. Subcell Biochem 62:327-41
Gamper, Armin M; Choi, Serah; Matsumoto, Yoshihiro et al. (2012) ATM protein physically and functionally interacts with proliferating cell nuclear antigen to regulate DNA synthesis. J Biol Chem 287:12445-54
Goula, Agathi-Vasiliki; Pearson, Christopher E; Della Maria, Julie et al. (2012) The nucleotide sequence, DNA damage location, and protein stoichiometry influence the base excision repair outcome at CAG/CTG repeats. Biochemistry 51:3919-32

Showing the most recent 10 out of 30 publications