In both budding and fission yeast, a large number of ribonucleotides are incorporated into DNA during replication by the major replicative polymerases (Pols α, δ, and ε). They are subsequently removed by RNase H2-dependent repair, which if defective leads to replication stress and genome instability. To extend these studies to humans, where an RNase H2 defect results in an autoimmune disease, here we compare the ability of human and yeast Pol δ to incorporate, proofread, and bypass ribonucleotides during DNA synthesis. In reactions containing nucleotide concentrations estimated to be present in mammalian cells, human Pol δ stably incorporates one rNTP for approximately 2,000 dNTPs, a ratio similar to that for yeast Pol δ. This result predicts that human Pol δ may introduce more than a million ribonucleotides into the nuclear genome per replication cycle, an amount recently reported to be present in the genome of RNase H2-defective mouse cells. Consistent with such abundant stable incorporation, we show that the 3´-exonuclease activity of yeast and human Pol δ largely fails to edit ribonucleotides during polymerization. We also show that, like yeast Pol δ, human Pol δ pauses as it bypasses ribonucleotides in DNA templates, with four consecutive ribonucleotides in a DNA template being more problematic than single ribonucleotides. In conjunction with recent studies in yeast and mice, this ribonucleotide incorporation may be relevant to impaired development and disease when RNase H2 is defective in mammals. As one tool to investigate ribonucleotide incorporation by Pol δ in human cells, we show that human Pol δ containing a Leu606Met substitution in the polymerase active site incorporates 7-fold more ribonucleotides into DNA than does wild type Pol δ.
To address the biochemical mechanisms underlying the coordination between the various proteins required for nucleotide excision repair (NER), we employed the immobilized template system. Using either wild-type or mutated recombinant proteins, we identified the factors involved in the NER process and showed the sequential comings and goings of these factors to the immobilized damaged DNA. Firstly, we found that PCNA and RF-C arrival requires XPF 5 0 incision. Moreover, the positioning of RF-C is facilitated by RPA and induces XPF release. Concomitantly, XPG leads to PCNA recruitment and stabilization. Our data strongly suggest that this interaction with XPG protects PCNA and Pold from the effect of inhibitors such as p21. XPG and RPA are released as soon as Pold is recruited by the RF-C/PCNA complex. Finally, a ligation system composed of FEN1 and Ligase I can be recruited to fully restore the DNA. In addition, using XP or trichothiodystrophy patient-derived cell extracts, we were able to diagnose the biochemical defect that may prove to be important for therapeutic purposes.
The actin filament-associated protein and Src-binding partner, AFAP-110, is an adaptor protein that links signaling molecules to actin filaments. AFAP-110 binds actin filaments directly and multimerizes through a leucine zipper motif. Cellular signals downstream of Src 527F can regulate multimerization. Here, we determined recombinant AFAP-110 (rAFAP-110)-bound actin filaments cooperatively, through a lateral association. We demonstrate rAFAP-110 has the capability to cross-link actin filaments, and this ability is dependent on the integrity of the carboxy terminal actin binding domain. Deletion of the leucine zipper motif or PKC phosphorylation affected AFAP-110's conformation, which correlated with changes in multimerization and increased the capability of rAFAP-110 to cross-link actin filaments. AFAP-110 is both a substrate and binding partner of PKC. On PKC activation, stress filament organization is lost, motility structures form, and AFAP-110 colocalizes strongly with motility structures. Expression of a deletion mutant of AFAP-110 that is unable to bind PKC blocked the effect of PMA on actin filaments. We hypothesize that upon PKC activation, AFAP-110 can be cooperatively recruited to newly forming actin filaments, like those that exist in cell motility structures, and that PKC phosphorylation effects a conformational change that may enable AFAP-110 to promote actin filament cross-linking at the cell membrane.
Common fragile sites (CFS) are hotspots of chromosomal breakage, and CFS breakage models involve perturbations of DNA replication. Here, we analyzed the contribution of specific repetitive DNA sequence elements within CFS to the inhibition of DNA synthesis by replicative and specialized DNA polymerases. The efficiency of in vitro DNA synthesis was quantitated using templates corresponding to regions within FRA16D and FRA3B harboring AT-rich microsatellite and quasi-palindrome (QP) sequences. QPs were predicted to form stems of ~75-100% self homology, separated by 3-9 bases of intervening sequences. Analysis of DNA synthesis progression by human DNA polymerase δ (Pol δ) demonstrated significant synthesis perturbation both at [A]n and [TA]n repeats in a length-dependent manner, and at short (<40 basepairs) QP sequences. DNA synthesis by the Y-family polymerase κ was significantly more efficient than Pol δ through both types of repetitive elements. Using DNA trap experiments, we show that Pol δ pauses within CFS sequences are sites of enzyme dissociation, and dissociation was observed in the presence of RFC-loaded PCNA. We propose that enrichment of microsatellite and QP elements at CFS regions contributes to fragility by perturbing replication through multiple mechanisms, including replicative DNA polymerase pausing and dissociation. Our finding that Pol δ dissociates at specific CFS sequences is significant, since dissociation of the replication machinery and inability to efficiently recover the replication fork can lead to fork collapse and/or formation of double-strand breaks in vivo. Our biochemical studies also extend the potential involvement of Y-family polymerases in CFS maintenance to include polymerase κ.
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