title: The C-terminal domain of HU-related histone-like protein Hlp from Mycobacterium smegmatis mediates DNA end-joining.
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, K d = 0.037 +/- 0.001 nM, compared to an Hlp variant without the C-terminal repeats, K d = 2.5 +/- 0.1 nM and the isolated C-terminal repeat domain, K d = 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.
authors.affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803, USA. agrove@lsu.edu
authors.forename: Anirban
authors.initials: A
authors.lastname: Mukherjee
authors.forename: Gargi
authors.initials: G
authors.lastname: Bhattacharyya
authors.forename: Anne
authors.initials: A
authors.lastname: Grove
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D003080:Cold Temperature; D004260:DNA Repair / Q000502:physiology; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D020102:Mycobacterium smegmatis / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D015854:Up-Regulation
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012329:RNA, Bacterial; C040330:histone-like protein HU, bacteria