Replication protein A 32 interacts through a similar binding interface with TIPIN, XPA, and UNG2

Ali, Seikh Imtiaz; Shin, Jeong Hyun; Bae, Sung-Hun; Kim, Byoungkook; Choi, Byong‐Seok

doi:10.1016/j.biocel.2010.04.011

Cited by 27 publications

(30 citation statements)

References 32 publications

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“…GST-pull-down assays confirmed the previously reported interaction between the Tim-Tipin complex and RPA (Supplementary Figure S1E) (3,14,37). The Tim-Tipin-RPA mixture showed a homogenic complex formation by native polyacrylamide gel electrophoresis (Figure 1A).…”

Section: Resultssupporting

confidence: 87%

“…In turn, RPA provides a binding platform for additional factors during DNA repair (35,36). These include Tim-Tipin (3,37), and the DNA repair factors XPA (xeroderma pigmentosum complementation group A protein) (38), UNG2 (uracyl DNA glycosylase-2) (39) and RAD52 (40), which are reported to bind to the WH domain of the RPA32 subunit. Tim-Tipin cooperate with RPA to assure the structural integrity of the replication fork, however, the nature of this interaction is elusive.…”

Section: Introductionmentioning

confidence: 99%

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Architecture and ssDNA interaction of the Timeless-Tipin-RPA complex

Witosch

Wolf

Mizuno

2014

Nucleic Acids Research

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The Timeless-Tipin (Tim-Tipin) complex, also referred to as the fork protection complex, is involved in coordination of DNA replication. Tim-Tipin is suggested to be recruited to replication forks via Replication Protein A (RPA) but details of the interaction are unknown. Here, using cryo-EM and biochemical methods, we characterized complex formation of Tim-Tipin, RPA and single-stranded DNA (ssDNA). Tim-Tipin and RPA form a 258 kDa complex with a 1:1:1 stoichiometry. The cryo-EM 3D reconstruction revealed a globular architecture of the Tim-Tipin-RPA complex with a ring-like and a U-shaped domain covered by a RPA lid. Interestingly, RPA in the complex adopts a horse shoe-like shape resembling its conformation in the presence of long ssDNA (>30 nucleotides). Furthermore, the recruitment of the Tim-Tipin-RPA complex to ssDNA is modulated by the RPA conformation and requires RPA to be in the more compact 30 nt ssDNA binding mode. The dynamic formation and disruption of the Tim-Tipin-RPA-ssDNA complex implicates the RPA-based recruitment of Tim-Tipin to the replication fork.

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Section: Resultssupporting

confidence: 87%

Section: Introductionmentioning

confidence: 99%

Architecture and ssDNA interaction of the Timeless-Tipin-RPA complex

Witosch

Wolf

Mizuno

2014

Nucleic Acids Research

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“…The NMR titration results agree well with observations from structural comparison. In another previous NMR-based study of Tipin peptide binding to RPA32C, chemical shift perturbations for those residues located in the a1/ a2 loop have also been observed, but the data interpretation and the built structural model are not accurate [21].…”

Section: Molecular Basis Of Smarcal1 Peptide Recognitionmentioning

confidence: 94%

Structure of RPA32 bound to the N‐terminus of SMARCAL1 redefines the binding interface between RPA32 and its interacting proteins

Xie

Jakoncic

et al. 2014

The FEBS Journal

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, Tipin, UNG2 and XPA. We have solved the high-resolution crystal structure of RPA32 C-terminal domain (RPA32C) in complex with a 26-amino-acid peptide derived from the N-terminus of SMARCAL1 (SMARCAL1N). The RPA32C-SMARCAL1N structure reveals a 1 : 1 binding stoichiometry and displays a well-ordered binding interface. SMARCAL1N adopts a long a-helical conformation with the highly conserved 11 residues aligned on one face of the a-helix showing extensive interactions with the RPA32C domain. Extensive mutagenesis experiments were performed to corroborate the interactions observed in crystal structure. Moreover, the a1/a2 loop of the RPA32C domain undergoes a conformational rearrangement upon SMARCAL1N binding. NMR study has further confirmed that the RPA32C-SMARCAL1N interaction induces conformational changes in RPA32C. Isothermal titration calorimetry studies have also demonstrated that the conserved a-helical motif defined in the current study is required for sufficient binding of RPA32C. Taken together, our study has provided convincing structural information that redefines the common recognition pattern shared by RPA32C interacting proteins. DatabaseThe atomic coordinates of RPA32C in complex with 26-aa SMARCAL1 (SMARCAL1N) peptide have been deposited at the Protein Data Bank with accession code 4MQV. Structured digital abstractRPA32 and SMARCAL1 bind by isothermal titration calorimetry (1,2,3,4,5,6,7,8,9) RPA32 and SMARCAL1 bind by molecular sieving (View interaction) RPA32 and SMARCAL1 bind by x-ray crystallography (View interaction) Tipin and RPA32 bind by isothermal titration calorimetry (1, 2) RPA32 and UNG2 bind by isothermal titration calorimetry (1, 2, 3) SMARCAL1 and RPA32 bind by nuclear magnetic resonance (View interaction) UNG2 and RPA32 bind by nuclear magnetic resonance (View interaction) Tipin and RPA32 bind by nuclear magnetic resonance (View interaction)Abbreviations ITC, isothermal titration calorimetry; NER, nucleotide excision repair; RPA, replication protein A; RPA32C, RPA32 C-terminal domain; SIOD, Schimke immuno-osseous dysplasia; Tipin, Timeless-interacting protein; UNG2, uracil-DNA glycosylase; XPA, xeroderma pigmentosum complementation group A.

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“…UNG2 is a base excision repair enzyme that participates in the cellular mechanisms for the specific removal of uracil residues from DNA resulting from misincorporation of dUTPs during replication or cytosine deamination (42). Therefore, the role of UNG2 in DNA repair at the replication fork during chromosomal replication is well established, since UNG2 contains determinants required for interactions with proliferating cell nuclear antigen (PCNA) and the 32-kDa subunit (RPA2) of replication protein A (RPA) (1,14,19,28,30). In addition, UNG2 plays a specific role in somatic hypermutations and class-switch recombination (CSR) at the immunoglobulin locus of B lymphocytes (42).…”

mentioning

confidence: 99%

Recruitment of the Nuclear Form of Uracil DNA Glycosylase into Virus Particles Participates in the Full Infectivity of HIV-1

Guenzel

Hérate

Rouzic

et al. 2012

J Virol

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The HIV-1 Vpr protein participates in the early steps of the virus life cycle by influencing the accuracy of reverse transcription. This role of Vpr was related to the recruitment of the nuclear form of the uracil DNA glycosylase (UNG2) enzyme into virus particles, but several conflicting findings have been reported regarding the role of UNG2 encapsidation on viral infectivity. Here, we report that the catalytic activity of UNG2 was not required for influencing HIV-1 mutation, and this function of UNG2 was mapped within a 60-amino-acid domain located in the N-terminal region of the protein required for direct interaction with the p32 subunit of the replication protein A (RPA) complex. Importantly, enforced recruitment of overexpressed UNG2 into virions resulted in a net increase of virus infectivity, and this positive effect on infectivity was also independent of the UNG2 enzymatic activity. In contrast, virus infectivity and replication, as well as the efficiency of the viral DNA synthesis, were significantly reduced when viruses were produced from cells depleted of either endogenous UNG2 or RPA p32. Taken together, these results demonstrate that incorporation of UNG2 into virions has a positive impact on HIV-1 infectivity and replication and positively influences the reverse transcription process through a nonenzymatic mechanism involving the p32 subunit of the RPA complex.

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Replication protein A 32 interacts through a similar binding interface with TIPIN, XPA, and UNG2

Cited by 27 publications

References 32 publications

Architecture and ssDNA interaction of the Timeless-Tipin-RPA complex

Architecture and ssDNA interaction of the Timeless-Tipin-RPA complex

Structure of RPA32 bound to the N‐terminus of SMARCAL1 redefines the binding interface between RPA32 and its interacting proteins

Recruitment of the Nuclear Form of Uracil DNA Glycosylase into Virus Particles Participates in the Full Infectivity of HIV-1

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