Abstract
In mammalian cells, the nucleosome-binding protein HMGN1 (high mobility group N1) affects the structure and function of chromatin and plays a role in repair of damaged DNA. HMGN1 affects the interaction of DNA repair factors with chromatin and their access to damaged DNA; however, not all of the repair factors affected have been identified. Here, we report that HMGN1 affects the self-poly(ADP-ribosyl)ation (i.e., PARylation) of poly(ADP-ribose) polymerase-1 (PARP-1), a multifunctional and abundant nuclear enzyme known to recognize DNA lesions and promote chromatin remodeling, DNA repair, and other nucleic acid transactions. The catalytic activity of PARP-1 is activated by DNA with a strand break, and this results in self-PARylation and PARylation of other chromatin proteins. Using cells obtained from Hmgn1(-/-) and Hmgn1(+/+) littermate mice, we find that in untreated cells, loss of HMGN1 protein reduces PARP-1 self-PARylation. A similar result was obtained after MMS treatment of these cells. In imaging experiments after low energy laser-induced DNA damage, less PARylation at lesion sites was observed in Hmgn1(-/-) than in Hmgn1(+/+) cells. The HMGN1 regulation of PARP-1 activity could be mediated by direct protein-protein interaction as HMGN1 and PARP-1 were found to interact in binding assays. Purified HMGN1 was able to stimulate self-PARylation of purified PARP-1, and in experiments with cell extracts, self-PARylation was greater in Hmgn1(+/+) than in Hmgn1(-/-) extract. The results suggest a regulatory role for HMGN1 in PARP-1 activation.
Highlights
HMGN1 affects the interaction of DNA repair factors with chromatin
The results confirmed expression of other Base excision repair (BER) factors including poly(ADP-ribose) polymerase-1 (PARP-1), APE1, pol , XRCC1, and DNA ligase I; the expression level of APE1 and XRCC1 appeared to be slightly higher in Hmgn1Ϫ/Ϫ cells, but the other proteins were expressed at similar levels in both cell lines (Fig. 1A)
We examined self-PARylation of PARP-1 as a function of HMGN1 expression in mouse fibroblasts in culture
Summary
HMGN1 affects the interaction of DNA repair factors with chromatin. Results: We identified a functional interaction between HMGN1 and PARP-1 in mouse fibroblast cells. The nucleosome-binding protein HMGN1 (high mobility group N1) affects the structure and function of chromatin and plays a role in repair of damaged DNA. Poly(ADP-ribose) polymerase 1 (PARP-1) is an abundant nuclear enzyme that has other activities such as chromatin structure modulation, transcriptional co-regulation, and genomic insulator function (30 –32). In addition to a role in BER, PARylation of PARP-1 plays various roles in other cellular processes, including chromatin modification, transcription, and cell death pathways. We compared the self-PARylation level of PARP-1 in Hmgn1ϩ/ϩ and Hmgn1Ϫ/Ϫ cells and found that it was substantially lower in the HMGN1 null cells This decrease in self-PARylation of PARP-1 may be due to limited access to DNA lesions in the absence of HMGN1 and could be due to a direct effect of HMGN1 on PARP-1 activity. The finding of an interaction between HMGN1 and PARP1 is discussed
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