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 Citations (Scopus)

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

Fingerprint

Mycobacterium smegmatis
Joining
Histones
DNA
Terminal Repeat Sequences
Proteins
Transcription
DNA Ligases
DNA Probes
Electrophoretic mobility
Electrophoretic Mobility Shift Assay
DNA Replication
Mycobacterium tuberculosis
Genetic Recombination
Shock
Up-Regulation
Assays
Peptides

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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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.",
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The C-terminal domain of HU-related histone-like protein Hlp from Mycobacterium smegmatis mediates DNA end-joining. / Mukherjee, Anirban; Bhattacharyya, Gargi; Grove, Anne.

In: Biochemistry, Vol. 47, No. 33, 19.08.2008, p. 8744-8753.

Research output: Contribution to journalArticle

TY - JOUR

T1 - The C-terminal domain of HU-related histone-like protein Hlp from Mycobacterium smegmatis mediates DNA end-joining

AU - Mukherjee, Anirban

AU - Bhattacharyya, Gargi

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N2 - 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.

AB - 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.

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