Interactions in the Error-prone Postreplication Repair Proteins hREV1, hREV3, and hREV7

Abstract

Most mutations after DNA damage in yeast Saccharomyces cerevisiae are induced by error-prone translesion DNA synthesis employing scRev1 and DNA polymerase that consists of scRev3 and scRev7 proteins. Recently, the human REV1 (hREV1) and REV3 (hREV3) genes were identified, and their products were revealed to be involved in UV-induced mutagenesis, as observed for their yeast counterparts. Human REV7 (hREV7) was also cloned, and its product was found to interact with hREV3, but the biological function of hREV7 remained unknown. We report here the analyses of precise interactions in the human REV proteins. The interaction between hREV1 and hREV7 was identified by the yeast two-hybrid library screening using a bait of hREV7, which was confirmed by in vitro and in vivo binding assays. The homodimerization of hREV7 was also detected in the two-hybrid analysis. In addition, the precise domains for interaction between hREV7 and hREV1 or hREV3 and for hREV7 homodimerization were determined. Although hREV7 interacts with both hREV1 and hREV3, a stable complex formation of the three proteins was undetectable in vitro. These findings suggest the possibility that hREV7 might play an important role in regulating the enzymatic activities of hREV1 and hREV3 for mutagenesis in response to DNA damage.An error-free DNA replication system is required to pass accurate genetic information on to the next generation. However, various kinds of DNA damage induced by endogenous and exogenous factors impair this replication ability and cause genetic alterations, resulting in cancer predisposition (1). Cells have excellent systems for avoiding these genetic alterations by removing and repairing the damaged lesions before DNA replication for maintaining the genetic stability of the organism; these systems include base excision repair, nucleotide excision repair, mismatch repair, and recombination repair (2, 3). If a lesion on template DNA escapes these repair systems, a polymerase may stall at this point and start synthesis again downstream, resulting in a single strand gap in the DNA, which can be repaired by postreplication repair. Usually, recombination repair in postreplication repair can fix this gap without base substitution, but when this repair does not happen, DNA synthesis by a bypass formation across the lesion called translesion synthesis (TLS) 1 may take place to fill the gap. This TLS may be held in the last resort for DNA repair because mutations can be induced during this step (for reviews, see Refs. 4 -6).In budding yeast Saccharomyces cerevisiae, the scRAD30 gene, the product of which is DNA polymerase , is involved in the error-free TLS that can replicate DNA through cis-syn thymine-thymine (T-T) dimer in an error-free manner (7-10), whereas the scREV1, scREV3, and scREV7 genes are involved in the error-prone TLS that frequently induces mutations at the damaged lesions (for reviews, see . It is known that most mutations induced after UV irradiation are caused by the products of these three REV genes. scRev1 protein is a ter...

DNA polymerase zeta (pol ζ) in higher eukaryotes

“…A fragment of human REV3L (amino acid residues 1776-2044) interacts with full-length REV7 as shown by yeast two-hybrid assays and by co-immunoprecipitation of polypeptides transiently expressed in HeLa cells [37]. Yeast two-hybrid assays show that a region of human REV7 (amino acids 21-155) interacts with human REV3L and REV1 [37]. However, an interaction between human REV7 and full-length human REV3L has not yet been demonstrated experimentally.…”

“…The mammalian REV1-REV7 interaction involves the C-terminal region (amino acids 1 130-1 251) of REV1 [37,39,40]. This Cterminal region of mammalian REV1 is important for inter- BRCT UBM npg acting with many other translesion synthesis polymerases.…”

“…(D) By a yet undefined mechanism, the lesion bypass complex dissociates from the template, Ub-PCNA is removed and the normal DNA replication polymerases reassociate and continue genome replication. [36][37][38][39][40].…”

The Vital Role of Polymerase ζ and REV1 in Mutagenic, but Not Correct, DNA Synthesis across Benzo[a]pyrene-dG and Recruitment of Polymerase ζ by REV1 to Replication-stalled Site

“…This enzyme activity plays a role in TLS across certain classes of lesions (10,11). REV1 also plays a noncatalytic role in TLS (12) and physically interacts with the other Y family polymerases (13,14) and the REV7 subunit of pol (15,16). The biological significance of these interactions remains to be determined.…”

Translesion synthesis in mammalian cells

“…Rev1 interacts with polη, ι, κ and Rev7, in all cases via the same domain contained in its C-terminal 150 aa [32][33][34]. It should also be borne in mind that PCNA is a homotrimer, and the available evidence suggests that ubiquitination is an all or nothing process, ie that all three monomers become ubiquitinated in one trimer [25,31].…”