Abstract
Single-stranded DNA binding proteins (SSBs) are vital in all organisms. SSBs of Escherichia coli (EcoSSB) and Mycobacterium tuberculosis (MtuSSB) are homotetrameric. The N-terminal domains (NTD) of these SSBs (responsible for their tetramerization and DNA binding) are structurally well defined. However, their C-terminal domains (CTD) possess undefined structures. EcoSSB NTD consists of β1-β1′-β2-β3-α-β4-β451-β452-β5 secondary structure elements. MtuSSB NTD includes an additional β-strand (β6) forming a novel hook-like structure. Recently, we observed that MtuSSB complemented an E. coli Δssb strain. However, a chimeric SSB (mβ4-β5), wherein only the terminal part of NTD (β4-β5 region possessing L45 loop) of EcoSSB was substituted with that from MtuSSB, failed to function in E. coli in spite of its normal DNA binding and oligomerization properties. Here, we designed new chimeras by transplanting selected regions of MtuSSB into EcoSSB to understand the functional significance of the various secondary structure elements within SSB. All chimeric SSBs formed homotetramers and showed normal DNA binding. The mβ4-β6 construct obtained by substitution of the region downstream of β5 in mβ4-β5 SSB with the corresponding region (β6) of MtuSSB complemented the E. coli strain indicating a functional interaction between the L45 loop and the β6 strand of MtuSSB.
Highlights
Single-stranded DNA binding protein (SSB) binds singlestranded DNA in a sequence independent manner during major DNA transactions such as DNA replication, repair and recombination [1,2,3,4,5]
Experimental rationale and generation of SSB chimeras The N-terminal domain of EcoSSB is defined by b1-b19-b2-b3
A notable difference seen at the level of tetramerization of MtuSSB is the presence of a clamp like structure formed by the b6 strand of the mycobacterial SSB [19]
Summary
Single-stranded DNA binding protein (SSB) binds singlestranded DNA in a sequence independent manner during major DNA transactions such as DNA replication, repair and recombination [1,2,3,4,5]. Besides their crucial function in DNA transactions, they protect transiently generated single-stranded DNA (ssDNA) from nucleases or chemical attacks [6]. While all the four subunits bind to ,56 or ,65 nucleotides in a limited cooperative manner known as SSB56 or SSB65 modes, respectively [13,14,15,16]
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