The C-terminal domain of HU-related histone-like protein Hlp from Mycobacterium smegmatis mediates DNA end-joining

Anirban Mukherjee, Gargi Bhattacharyya, Anne Grove

Research output: Contribution to journalArticle

24 Scopus citations

Abstract

Histone-like proteins (such as HU, H-NS, and Fis) participate in nucleoid organization and in DNA replication, recombination, and transcription. Cold shock and anoxia upregulates a homologue of HU (Hlp) in Mycobacterium smegmatis, the nonpathogenic model of Mycobacterium tuberculosis. We show using electrophoretic mobility shift assays that Hlp, which in addition to the HU fold has a basic C-terminal tail containing multiple PAKK and PAAK repeats, has very high affinity for DNA. The affinity of Hlp for 76 bp linear DNA is higher, Kd = 0.037 ± 0.001 nM, compared to an Hlp variant without the C-terminal repeats, Kd = 2.5 ± 0.1 nM and the isolated C-terminal repeat domain, Kd = 0.8 ± 0.2 nM, where K d in all cases reflects an aggregate affinity for the DNA probes, not the affinity for binding to a single site. Hlp lacking the entire C-terminal domain binds DNA only poorly. These data indicate that both Hlp domains contribute to high-affinity DNA binding. Hlp promotes DNA end-joining in the presence of T4 DNA ligase, and this property is mediated by the C-terminal repeats. At <100 nM concentration, Hlp represses transcription by T7 RNA polymerase in vitro whereas the individual N- and C-terminal domains do not, even when present together. Notably, while DNA end-joining can be achieved by the isolated C-terminal domain, transcriptional repression requires for both domains to be present on a single polypeptide. Given the low cellular concentration of Hlp, our data suggest that its primary functional role may be in DNA-dependent responses to environmental stress rather than in nucleoid organization.

Original languageEnglish (US)
Pages (from-to)8744-8753
Number of pages10
JournalBiochemistry
Volume47
Issue number33
DOIs
StatePublished - Aug 19 2008

All Science Journal Classification (ASJC) codes

  • Biochemistry

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