The Catalytic Activity of Human REV1 on Undamaged and Damaged DNA.

Eukaryotic REV1 serves as a scaffold protein for the coordination of DNA polymerases during DNA translesion synthesis. Besides this structural role, REV1 is a Y-family DNA polymerase with its own distributive deoxycytidyl transferase activity. However, data about the accuracy and efficiency of DNA synthesis by REV1 in the literature are contrasting. Here, we expressed and purified the full-length human REV1 from Saccharomyces cerevisiae and characterized its activity on undamaged DNA and a wide range of damaged DNA templates. We demonstrated that REV1 carried out accurate synthesis opposite 8-oxoG and O6-meG with moderate efficiency. It also replicated thymine glycol surprisingly well in an error-prone manner, but was blocked by the intrastrand 1,2-GG cisplatin crosslink. By using the 1,N6-ethenoadenine and 7-deaza-adenine lesions, we have provided biochemical evidence of the importance for REV1 functioning of the Hoogsteen face of template A, the second preferable template after G.

Impaired function of rDNA transcription initiation machinery leads to derepression of ribosomal genes with insertions of R2 retrotransposon.

Eukaryotic genomes harbor hundreds of rRNA genes, many of which are transcriptionally silent. However, little is known about selective regulation of individual rDNA units. In Drosophila melanogaster, some rDNA repeats contain insertions of the R2 retrotransposon, which is capable to be transcribed only as part of pre-rRNA molecules. rDNA units with R2 insertions are usually inactivated, although R2 expression may be beneficial in cells with decreased rDNA copy number. Here we found that R2-inserted rDNA units are enriched with HP1a and H3K9me3 repressive mark, whereas disruption of the heterochromatin components slightly affects their silencing in ovarian germ cells. Surprisingly, we observed a dramatic upregulation of R2-inserted rRNA genes in ovaries lacking Udd (Under-developed) or other subunits (TAF1b and TAF1c-like) of the SL1-like complex, which is homologues to mammalian Selective factor 1 (SL1) involved in rDNA transcription initiation. Derepression of rRNA genes with R2 insertions was accompanied by a reduction of H3K9me3 and HP1a enrichment. We suggest that the impairment of the SL1-like complex affects a mechanism of selective activation of intact rDNA units which competes with heterochromatin formation. We also propose that R2 derepression may serve as an adaptive response to compromised rRNA synthesis.