A wild-type DNA ligase I gene is expressed in Bloom's syndrome cells.

Abstract

Alteration of DNA ligase I activity is a consistent biochemical feature of Bloom's syndrome (BS) cells.DNA ligase I activity in BS cells either is reduced and abnormally thermolabile or is present in an anamolously dimeric form. To assess the role of DNA ligase function in the etiology of BS, we have cloned the DNA ligase I cDNA from normal human cells by a PCR strategy using degenerate oligonucleotide primers based on conserved regions of the Saccharomyces cerevisiae and Schizosaccharomyces pombe DNA ligase genes.Human DNA ligase I cDNAs from normal and BS cells complemented a S. cerevisiae DNA ligase mutation, and protein extracts prepared from S. cerevisiae transformants expressing normal and BS cDNA contained comparable levels of DNA ligase I activity. DNA sequencing and Northern blot analysis of DNA ligase I expression in two BS human fibroblast lines representing each of the two aberrant DNA ligase I molecular phenotypes demonstrated that this gene was unchanged in BS cells. Thus, another factor may be responsible for the observed reduction in DNA ligase I activity associated with this chromosomal breakage syndrome.To assess the molecular basis of this disease, we have cloned the cDNA corresponding to the human DNA ligase I gene expressed in normal cells and from two BS fibroblast lines, GM8505 and GM5289. The DNA ligase I gene was isolated by a PCR strategy employing degenerate oligonucleotides based on conserved regions of the DNA ligase genes of Saccharomyces cerevisiae and Schizosaccharomyces pombe (15). A complete DNA sequence analysis from two BS cell lines revealed that each BS DNA ligase I gene was indistinguishable from the wild-type gene. A single 3.1-kilobase (kb) DNA ligase I mRNA was detected at normal steady-state levels in BS cells by Northern blot analysis. Also, the GM8505 DNA ligase I gene was able to complement the S. cerevisiae cdc9 DNA ligase mutation. Given the reduction in DNA ligase I activity in BS cells, these results suggest that a factor which modulates DNA ligase I activity in normal cells is defective in BS. Alternatively, a defect in a modifying activity that acts upon DNA ligase I and some DNA-repair enzymes may account for the altered repair phenotypes in BS cells.

Replication Protein A and the Mre11·Rad50·Nbs1 Complex Co-localize and Interact at Sites of Stalled Replication Forks

“…Recent data suggest that the MRN complex functions as a damage sensor upstream of ATM/ATR activation in addition to acting as an effector downstream of ATM/ATR in double strand break repair and cell cycle checkpoints (17)(18)(19)(20). Although it is known that the MRN complex binds to DNA ends, there is no clearly defined mechanism by which the MRN complex recognizes other types of DNA damage.…”

Dna Repair in Humans

“…Reports of a ligase I defect in BS syndrome have not been confirmed (124,148). Similarly, claims of defects in uracil glycosylase or SOD have been discarded based on the fact that the chromosome 15 , which complements BS, does not carry the genes for these enzymes (120).…”

Human DNA Ligase I Interacts with and Is Targeted for Degradation by the DCAF7 Specificity Factor of the Cul4-DDB1 Ubiquitin Ligase Complex

“…In humans, there are three genes that encode DNA ligases, LIG1, LIG3, and LIG4 (1)(2)(3). There is compelling cell biology, biochemical, and molecular genetic evidence indicating that the DNA ligase encoded by the mammalian LIG1 gene, DNA ligase I (LigI), 4 plays the predominant role in DNA replication. The expression of the LIG1 gene is increased when quiescent cells are induced to proliferate (4).…”

“…There is compelling cell biology, biochemical, and molecular genetic evidence indicating that the DNA ligase encoded by the mammalian LIG1 gene, DNA ligase I (LigI), 4 plays the predominant role in DNA replication. The expression of the LIG1 gene is increased when quiescent cells are induced to proliferate (4). In addition, LigI physically and functionally interacts with two key DNA replication protein complexes, proliferating cell nuclear antigen (PCNA) and replication factor C (RFC), and co-localizes with these factors in replication foci during S phase (5)(6)(7)(8).…”

“…The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. 4 The abbreviations used are: LigI, DNA ligase I; hLigI, human LigI; Cul4, cullin4; DDB1, DNA damage-specific DNA-binding protein 1; PCNA, proliferating cell nuclear antigen; FEN-1, flap endonuclease 1. …”