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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cancer Etiology Study Section (CE)
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Barski, Oleg
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University of New Mexico Health Sciences Center
Internal Medicine/Medicine
Schools of Medicine
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