Replicative Bypass Research Articles

Chromosome aberration formation and sister chromatid exchange in relation to DNA repair in human cells.

Apparent association between the ability to induce chromosome aberrations and sister chromatid exchanges and mutagenic-carcinogenic potential found in a variety of physical and chemical agents has led us to speculate that these cytogenetic changes might be reflection of DNA damage and repair and might provide induces of mutagenic changes. However, the mechanisms of their formation and their relation to DNA repair as well as the mechanism of their linking to mutation are by no means well understood. Studies in some human genetic mutant cells defective in their ability to repair DNA damage indicate, as a testable proposition, that sister chromatid exchanges and chromosome aberrations are cytological manifestations of replication-mediated dual-step repair pathways that are in operation to tolerate DNA damage when damage-bearing DNA enters and passes through semiconservative replication. The observations are also in line with idea that the majority of sister chromatid exchanges can arise when damage DNA attempts replication possibly by a process relating with the replicative bypass repair mechanisms such as those proposed by Fujiwara and Tatsumi [34] and Higgins et al. [54], while chromosome aberration formation and some fraction of sister chromatid exchanges are related with the post-replication repair processes which attempt to rescue damaged template post-replicationally by de novo synthesis or recombination type repair systems. The former sister chromatid exchange-relating process seems to link mutation to less extent, if any, than the latter process, which is caffeine sensitive and likely to be error-prone.

Further analysis of the replication bypass model for sister chromatid exchange.

The replication bypass model for sister chromatid exchange (SCE) proposed by Shafer is examined in detail. When applied through two cell cycles, the model predicts that potentially observable SCEs induced during one S phase will then be cancelled and rendered undetectable during the subsequent S phase. This aspect of replication bypass is inconsistent with the observation of twin SCEs in tetraploid cells. Furthermore, the model cannot account for the efficient induction of SCEs by non-cross-linking chemical agents.

DNA replication in ultraviolet light irradiated Chinese hamster cells: The nature of replicon inhibition and post-replication repair

DNA replication in ultraviolet light irradiated Chinese hamster cells was studied using techniques of DNA fiber autoradiography and alkaline sucrose sedimentation. Bidirectionally growing replicons are observed in the autoradiograms independent of the irradiation conditions. After a dose of 5 J/m 2 at 254 nm the rate of fork progression is the same as in unirradiated cells, while the rate of replication is reduced by 50%. After a dose of 10 J/m 2 the rate of fork progression is reduced 40%, while the replication rate is only 25% of normal. Therefore, at low doses of ultraviolet light irradiation, the inhibition of DNA replication is due to a reduction in the number of functioning replicons, while at higher doses the rate of fork progression is also slowed. Those replicons which no longer function after irradiation are blocked in fork movement rather than replicon initiation. After irradiation, pulse label is first incorporated into short nascent strands, the average size of which is approximately equal to the distance between pyrimidine dimers. Under conditions where post-replication repair occurs these short strands are eventually joined into larger pieces. Finally, the data show that slowing post-replication repair with caffeine does not slow fork movement. The results presented here support the post-replication repair model of “gapped synthesis” ( Rupp & Howard-Flanders, 1968) and rule out a major role for “replicative bypass” ( Higgins et al., 1976 ; Fujiwara & Tatsumi, 1976).

DOMINANT CHROMOSOMAL MUTATION BYPASSING CHROMOSOMAL GENES NEEDED FOR KILLER RNA PLASMID REPLICATION IN YEAST

Yeast strains carrying a double-stranded RNA plasmid of 1.4-1.7 x 10(6) daltons encapsulated in virus-like particles secrete a toxin that kills strains lacking this plasmid. The plasmid requires at least 24 chromosomal genes (pets, and mak1 through mak23) for its replication or maintenance. We have detected dominant Mendelian mutations (called KRB1 for killer replication bypass) that bypass two chromosomal genes, mak7 and pets, normally needed for plasmid replication. Strains mutant in mak7 and carrying the bypass mutation (mak7-1 KRB1) are isolated as frequent K(+)R(+) sectors of predominantly K(-)R( -) segregants from crosses of mak7-1 with a wild-type killer. All KRB1 mutations isolated in this way are inherited as single dominant centromere-linked chromosomal changes. They define a new centromere. KRB1 is not a translational suppressor. KRB1 strains contain a genetically normal killer plasmid and ds RNA species approximately the same in size and amount as do wild-type killers. Bypass of both mak7 and pets by one mutation suggests that these two genes are functionally related. Two properties of the inheritance of KRB1 indicate an unusually high reversion frequency: (1) Heat or cycloheximide (treatments known to cure strains of the wild-type killer plasmid) readily induce conversion of mak7-1 KRB1 strains from killers to nonkillers with concomitant disappearance of KRB1 as judged by further crosses, and (2) mating two strains of the type mak7-1 KRB1 with each other yields mostly 2 K(+)R(+): 2 K(-)R(-) segregation, although the same KRB1 mutation and the same killer plasmid are present in both parents.

Comparative studies on replicative bypass repair of UV damage to DNA in various lines of mammalian cells

Comparative studies on replicative bypass repair of UV damage to DNA in various lines of mammalian cells

Replication bypass model of sister chromatid exchanges and implications for Bloom's syndrome and Fanconi's anemia.

A model of the sister chromatid exchange (SCE) process is outlined as a replication mechanism to bypass DNA crosslinks. The model suggests that when normal bidirectional replication advances from both sides towards a crosslink along the two opposite parental strands, the complementary parental strand segments can be temporarily displaced at each contralateral 5' side from the crosslink. The free ends produced in this first step will be terminally aligned but will have opposite polarity. The second step of the bypass can, however, be completed by either of two rejoining processes--terminal ligation of the free ends via nascent Okazaki pieces or aberrant complementation by overlapping the free ends. This bypass mechanism (1) allows replication to continue past a crosslink leaving it intact but (2) results in the switching of parental strands and their attached incomplete nascent strands above and below the crosslink site producing an exchange between sister chromatids. This model is compatible with the findings of current SCE studies using the new BUDR/stain techniques as well as with previous autoradiographic studies. It also suggests that the chromatid breaks and deletions in Fanconi's Anemia represent a defect in step two of the replication bypass mechanism and that the high frequency of SCE's and quadriradials in Bloom's Syndrome represent the SCE overload effects of a defect in crosslink repair.

Replicative bypass repair of ultraviolet damage to DNA of mammalian cells: Caffeine sensitive and caffeine resistant mechanisms

Replicative bypass repair of UV damage to DNA was studied in wide variety of human, mouse and hamster cells in culture. Survival curve analysis revealed that in established cell lines (mouse L, Chinese hamster V79, HeLa S3 and SV40-transformed xeroderma pigmentosum (XP)), post-UV caffeine treatment potentiated cell killing by reducing the extrapolation number and mean lethal UV fluence ( Do). In the Do reduction as the result of random inactivation by caffeine of sensitive repair there were marked clonal differences among such cell lines, V79 being most sensitive to caffeine potentiation. However, other diploid cell lines (normal human, excision-defective XP and Syrian hamster) exhibited no obvious reduction in Do by caffeine. In parallel, alkaline sucrose sedimentation results showed that the conversion of initially smaller segments of DNA synthetized after irradiation with 10 J/m 2 to high-molecular-weight DNA was inhibited by caffeine in transformed XP cells, but not in the diploid human cell lines. Exceptionall, diploid XP variants had a retarded ability of bypass repair which was drastically prevented by caffeine, so that caffeine enhanced the lethal effect of UV. Neutral CsCl study on the bypass repair mechanism by use of bromodeoxyuridine for DNA synthesis on damaged template suggests that the pyrimidine dimer acts as a block to replication and subsequently it is circumvented presumably by a new process involving replicative bypassing following strand displacement, rather than by gap-filling de novo. This mechanism worked similarly in normal and XP cells, whether or not caffeine was present, indicating that excision of dimer is not always necessary. However, replicative became defective in XP variant and transformed XP cells when caffeine was present. It appears, therefore, that the replicative bypass repair process is either caffeine resistant or sensitive, depending on the cell type used, but not necessarily on the excision repair capability.

Contributions to railway statistics, in 1846, 1847, and 1848

In vitro replication assays for detection and quantification of bypass of UV-induced DNA photoproducts were used to compare the capacity of extracts prepared from different human cell lines to replicate past the cis,syn cyclobutane thymine dimer ([c,s]TT). The results demonstrated that neither nucleotide excision repair (NER) nor mismatch repair (MMR) activities in the intact cells interfered with measurements of bypass replication efficiencies in vitro. Extracts prepared from HeLa (NER- and MMR-proficient), xeroderma pigmentosum group A (NER-deficient), and HCT116 (MMR-deficient) cells displayed similar capacity for translesion synthesis, when the substrate carried the site-specific [c,s]TT on the template for the leading or the lagging strand of nascent DNA. Extracts from xeroderma pigmentosum variant cells, which lack DNA polymerase η, were devoid of bypass activity. Bypass-proficient extracts as a group (n=16 for 3 extracts) displayed higher efficiency (P=0.005) for replication past the [c,s]TT during leading strand synthesis (84±22%) than during lagging strand synthesis (64±13%). These findings are compared to previous results concerning the bypass of the (6–4) photoproduct [Biochemistry 40 (2001) 15215] and analyzed in the context of the reported characteristics of bypass DNA polymerases implicated in translesion synthesis of UV-induced DNA lesions. Models to explain how these enzymes might interact with the DNA replication machinery are considered. An alternative pathway of bypass replication, which avoids translesion synthesis, and the mutagenic potential of post-replication repair mechanisms that contribute to the duplication of the human genome damaged by UV are discussed.