Near-full-length REV3L appears to be a scarce maternal factor in Xenopus laevis eggs that changes qualitatively in early embryonic development.

REV3 is the catalytic subunit of DNA polymerase zeta (pol zeta), which is responsible for the damage-induced mutagenesis that arises during error-prone translesion synthesis in eukaryotes. The related REV3L genes in human and mouse encode proteins of approximately 350kDa, twice as large as yeast REV3, but full-length REV3L has not been identified in any vertebrate cell. We report that Xenopus laevisREV3L encodes a 352-kDa protein that has high overall amino acid sequence similarity to its mammalian counterparts, and, for the first time in a vertebrate species, we have detected putative REV3L polypeptides of 300 and 340kDa in X. laevis oocytes. Only the 300-kDa form is stored in eggs, where its concentration of about 65pM is much lower than those of other replication and repair proteins including the accessory pol zeta subunit REV7. In fertilized eggs, the levels of this polypeptide did not change until neurula; the larger 340-kDa form first appeared at stages after gastrula, suggesting a pattern of regulation during development. These observations indicate the existence of REV3L as a scarce protein, of approximately the full predicted size, whose level may impose severe constraints on the assembly of pol zeta in X. laevis.

Distinct roles of DNA polymerases delta and epsilon at the replication fork in Xenopus egg extracts.

DNA polymerases delta and epsilon (Poldelta and Polepsilon) are widely thought to be the major DNA polymerases that function in elongation during DNA replication in eukaryotic cells. However, the precise roles of these polymerases are still unclear. Here we comparatively analysed DNA replication in Xenopus egg extracts in which Poldelta or Polepsilon was immunodepleted. Depletion of either polymerase resulted in a significant decrease in DNA synthesis and accumulation of short nascent DNA products, indicating an elongation defect. Moreover, Poldelta depletion caused a more severe defect in elongation, as shown by sustained accumulation of both short nascent DNA products and single-stranded DNA gaps, and also by elevated chromatin binding of replication proteins that function more frequently during lagging strand synthesis. Therefore, our data strongly suggest the possibilities that Poldelta is essential for lagging strand synthesis and that this function of Poldelta cannot be substituted for by Polepsilon.