Dataset Information

Trans cooperativity by a split DNA recombinase: the central and catalytic domains of bacteriophage lambda integrase cooperate in cleaving DNA substrates when the two domains are not covalently linked.

ABSTRACT: Site-specific recombinases of the lambda-integrase family recognize and cleave their cognate DNA sites through cooperative binding to opposite sides of the DNA substrate by a C-terminal catalytic domain and a flexibly linked "core-binding" domain; regulation of this cleavage is achieved via the formation of higher-order complexes. We report that the core-binding domain of lambda-integrase is able to stimulate the activity of the catalytic domain even when the two domains are not linked. This trans stimulation is accomplished without significantly increasing the affinity of the catalytic domain for its DNA substrate. Moreover, we show that mutations in the DNA substrate can abrogate this effect while retaining specificity determinants for cleavage. Since the domains do not significantly interact directly, this finding implies that trans activation is achieved via the DNA substrate in a manner that may be mechanistically important in this and similar DNA binding and cleaving enzymes.

SUBMITTER: Subramaniam S

PROVIDER: S-EPMC2034338 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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Trans cooperativity by a split DNA recombinase: the central and catalytic domains of bacteriophage lambda integrase cooperate in cleaving DNA substrates when the two domains are not covalently linked.

Subramaniam Srisunder S Kamadurai Hari B HB Kamadurai Hari B HB Foster Mark P MP

Journal of molecular biology 20070419 2

Site-specific recombinases of the lambda-integrase family recognize and cleave their cognate DNA sites through cooperative binding to opposite sides of the DNA substrate by a C-terminal catalytic domain and a flexibly linked "core-binding" domain; regulation of this cleavage is achieved via the formation of higher-order complexes. We report that the core-binding domain of lambda-integrase is able to stimulate the activity of the catalytic domain even when the two domains are not linked. This tra ...[more]

PMID: 17531268

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Project description:The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5'-2-deoxyribose-5-phosphate lyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or an 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated -1 or -2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of -2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of -1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase within the non-homologous end joining pathway.

Dataset Information

Trans cooperativity by a split DNA recombinase: the central and catalytic domains of bacteriophage lambda integrase cooperate in cleaving DNA substrates when the two domains are not covalently linked.

Publications

Trans cooperativity by a split DNA recombinase: the central and catalytic domains of bacteriophage lambda integrase cooperate in cleaving DNA substrates when the two domains are not covalently linked.

Similar Datasets

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