Evolutionary conservation and DNA binding properties of the Ssh7 proteins fromSulfolobus shibatae |
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Authors: | Xulin Chen Rong Guo Li Huang Ray Hong |
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Institution: | (1) State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100080 Beijing, China;(2) Pomona College, 91711, CA, USA |
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Abstract: | The thermoacidophilic archaeonSulfolobus shibatae synthesizes a large amount of the 7-ku DNA binding proteins known as Ssh7. Our hybridization experiments showed that two
Ssh7-encoding genes existed in the genome of S.shibatae. These two genes, designatedssh7a andssh7b, have been cloned, sequenced and expressed inEscherichia coli. The two Ssh7 proteins differ only at three amino acid positions. In addition, thecis-regulatory sequences of thessh7a andssh7b genes are highly conserved. These results suggest the presence of a selective pressure to maintain not only the sequence
but also the expression of the two genes. We have also found that there are two genes encoding the 7-ku protein inSulfolobus solfataricus. Based on this and other studies, we suggest that the gene encoding the 7-ku protein underwent duplication before the separation
ofSulfolobus species. Binding of native Ssh7 and recombinant (r)Ssh7 to short duplex DNA fragments was analyzed by electrophoretic mobility
shift assays. Both native and recombinant forms of the protein behaved in a similar fashion in the assays, suggesting that
the interaction of Ssh7 with DNA is not affected either by specific lysine methylation found in the native Ssh7 proteins or
by the difference between the two Ssh7 isomers in amino acid sequence. Our data show that Ssh7 binds duplex DNA fragments
with a binding size of ∼ 6.6 base pairs and an apparent dissociation constant of (0.7–1.0) × 10-7 mol/L under the assay conditions employed in the present study. In addition, Ssh7 binds more tightly to negatively supercoiled
DNA than to linear or relaxed DNA. |
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Keywords: | hyperthermophilic archaea DNA binding proteins ssh 7a/ssh 7b genes protein-DNA interaction |
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