A RAD52-like single-stranded DNA binding protein affects mitochondrial DNA repair by recombination

ABSTRACT Disruption of cellular metabolic processes and usurpation of host proteins are hallmarks of herpesvirus lytic infection. Epstein-Barr virus (EBV) lytic replication is initiated by the immediate-early protein Zta. Zta is a multifunctional DNA binding protein that stimulates viral gene transcription, nucleates a replication complex at the viral origin of lytic replication, and inhibits cell cycle proliferation. To better understand these functions and identify cellular collaborators of Zta, we purified an epitope-tagged version of Zta in cells capable of supporting lytic replication. FLAG-tagged Zta was purified from a nuclear fraction using FLAG antibody immunopurification and peptide elution. Zta-associated proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by mass spectrometry. The Zta-associated proteins included members of the HSP70 family and various single-stranded DNA and RNA binding proteins. The nuclear replication protein A subunits (RPA70 and RPA32) and the human mitochondrial single-stranded DNA binding protein (mtSSB) were confirmed by Western blotting to be specifically enriched in the FLAG-Zta immunopurified complex. mtSSB coimmunoprecipitated with endogenous Zta during reactivation of EBV-positive Burkitt lymphoma and lymphoblastoid cell lines. Small interfering RNA depletion of mtSSB reduced Zta-induced lytic replication of EBV but had only a modest effect on transcription activation function. A point mutation in the Zta DNA binding domain (C189S), which is known to reduce lytic cycle replication, eliminated mtSSB association with Zta. The predominantly mitochondrial localization of mtSSB was shifted to partly nuclear localization in cells expressing Zta. Mitochondrial DNA synthesis and genome copy number were reduced by Zta-induced EBV lytic replication. We conclude that Zta interaction with mtSSB serves the dual function of facilitating viral and blocking mitochondrial DNA replication.

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Regulation of Deinococcus radiodurans RecA Protein Function via Modulation of Active and Inactive Nucleoprotein Filament States

Journal of Biological Chemistry ◽

10.1074/jbc.m113.459230 ◽

2013 ◽

Vol 288 (29) ◽

pp. 21351-21366 ◽

Cited By ~ 19

Author(s):

Khanh V. Ngo ◽

Eileen T. Molzberger ◽

Sindhu Chitteni-Pattu ◽

Michael M. Cox

Keyword(s):

Dna Repair ◽

Dna Binding ◽

Protein Function ◽

Deinococcus Radiodurans ◽

Binding Protein ◽

Reca Protein ◽

Optimal Growth ◽

Dna Binding Protein ◽

Single Stranded Dna ◽

Recombinational Dna Repair

The RecA protein of Deinococcus radiodurans (DrRecA) has a central role in genome reconstitution after exposure to extreme levels of ionizing radiation. When bound to DNA, filaments of DrRecA protein exhibit active and inactive states that are readily interconverted in response to several sets of stimuli and conditions. At 30 °C, the optimal growth temperature, and at physiological pH 7.5, DrRecA protein binds to double-stranded DNA (dsDNA) and forms extended helical filaments in the presence of ATP. However, the ATP is not hydrolyzed. ATP hydrolysis of the DrRecA-dsDNA filament is activated by addition of single-stranded DNA, with or without the single-stranded DNA-binding protein. The ATPase function of DrRecA nucleoprotein filaments thus exists in an inactive default state under some conditions. ATPase activity is thus not a reliable indicator of DNA binding for all bacterial RecA proteins. Activation is effected by situations in which the DNA substrates needed to initiate recombinational DNA repair are present. The inactive state can also be activated by decreasing the pH (protonation of multiple ionizable groups is required) or by addition of volume exclusion agents. Single-stranded DNA-binding protein plays a much more central role in DNA pairing and strand exchange catalyzed by DrRecA than is the case for the cognate proteins in Escherichia coli. The data suggest a mechanism to enhance the efficiency of recombinational DNA repair in the context of severe genomic degradation in D. radiodurans.

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