The importance of recombinations occuring in the process of DNA replication

In the process of DNA replication in Escherichia coli, the unequal crossing over occurs between DNA direct repeats in sister chromosomes. Such exchanges lead to the formation of tandem duplications and, therefore, to an increase of gene expression. It is proposed that non-homologous cohesion of sister chromosomes and unequal crossing over occur if the movement of replication fork is stopped. In the case of the unceasing movement of replication fork the homologous exchange between sister chromosomes occurs which provides their post-replication reparation.     

Mitotic inter-homologue junctions accumulate at damaged DNA replication forks in recQ mutants

Mitotic homologous recombination is utilised to repair DNA breaks using either sister chromatids or homologous chromosomes as templates. Because sister chromatids are identical, exchanges between sister chromatids have no consequences for the maintenance of genomic integrity unless they involve repetitive DNA sequences. Conversely, homologous chromosomes might differ in genetic content, and exchanges between homologues might lead to loss of heterozygosity and subsequent inactivation of functional genes. Genomic instability, caused by unscheduled recombination events between homologous chromosomes, is enhanced in the absence of RecQ DNA helicases, as observed in Bloom's cancer-prone syndrome. Here, we used two-dimensional gel electrophoresis to analyse budding yeast diploid cells that were modified to distinguish replication intermediates originating from each homologous chromosome. Therefore, these cells were suitable for analysing the formation of inter-homologue junctions. We found that Rad51-dependent DNA structures resembling inter-homologue junctions accumulate together with sister chromatid junctions at damaged DNA replication forks in recQ mutants, but not in the absence of Srs2 or Mph1 DNA recombination helicases. Inter-homologue joint molecules in recQ mutants are less abundant than sister chromatid junctions, but they accumulate with similar kinetics after origin firing under conditions of DNA damage. We propose that unscheduled accumulation of inter-homologue junctions during DNA replication might account for allelic recombination defects in recQ mutants.     

The function of recombinations occurring in the process of DNA replication in Escherichia coli

In a number of works dealing with the relationship between replication and recombination in bacteria, it is assumed that recombinations permit the replication forks to resume moving after having stopped at the damage sites of the template DNA. As an evidence for recombination occurring during DNA replication, the involvement in this process of proteins RuvABC and RecG, providing processing of the Holliday junctions after recombination, is considered. However, it has been shown that these proteins are not essential for resuming DNA synthesis after an exposure of bacteria to UV light. These data cast doubt on the necessity of recombination for reactivation of replication initiated in the oriC region. Studying recombination in tandem duplications in Escherichia coli showed that during replication, unequal crossing over occurs between direct DNA repeats of sister chromosomes. In wild strains, this crossing over results in tandem duplications, thereby enhancing the expression of certain genes. Thus, recombination of two types occurs during DNA replication: unequal crossing over leading to duplications and homologous exchange, responsible for post-replication DNA repair. The unequal exchange constitutes a component of SOS response of the cell to deterioration of the environment.     

Unequal crossing-over in Escherichia coli

Unequal crossing-over between sister chromosomes in the process of DNA replication in Escherichia coli leads to the formation of tandem duplications, thus enhancing the activity of certain genes. In conjugational matings between genetically marked E. coli strains, unequal crossing-over leads to the formation of heterozygous tandem duplications. Studying these duplications as model systems allowed the conclusion that unequal crossing-over between direct DNA repeats of sister chromosomes is the main pathway of the formation of selected recombinants in E. coli strains carrying duplications. This was inferred from the data on the segregation of homozygous diploid recombinants by heterozygous duplications. Unequal crossing-over between sister chromosomes occurs as adaptive exchange providing the survival of the greater part of bacterial cells on a selective medium. The known phenomenon of adaptive mutagenesis may also be a consequence of unequal exchanges at the level of DNA mononucleotide repeats.     

Unequal crossing over is the principal pathway of homologous recombination in tandem duplications of Escherichia coli

Homologous recombination between direct DNA repeats in tandem duplications usually leads to their dissociation. An even number of crossovers between two copies of a duplication should lead to the formation of diploid segregants, i.e., to the preservation of the duplication. However, in studies of the genotype of diploid segregants in heterozygous tandem duplications of Escherichia coli, it was shown that they arise by unequal exchanges between sister chromosomes rather than by intrachromosomal exchanges. Generally, these exchanges lead to the establishment of the homozygous state of (heterozygous) duplications. Since the available data suggest that the exchange between sister chromosomes may be coupled with DNA replication, it is supposed that unequal exchanges between direct DNA repeats occur in the process of DNA replication.     

Unequal Crossing Over Is the Principal Pathway of Homologous Recombination in Tandem Duplications of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Homologous recombination between direct DNA repeats in tandem duplications usually leads to their dissociation. An even number of crossovers between two copies of a duplication should lead to the formation of diploid segregants, i.e., to the preservation of the duplication. However, in studies of the genotype of diploid segregants in heterozygous tandem duplications of Escherichia coli, it was shown that they arise by unequal exchanges between sister chromosomes rather than by intrachromosomal exchanges. Generally, these exchanges lead to the establishment of the homozygous state of (heterozygous) duplications. Since the available data suggest that the exchange between sister chromosomes may be coupled with DNA replication, it is supposed that unequal exchanges between direct DNA repeats occur in the process of DNA replication.__________Translated from Genetika, Vol. 41, No. 8, 2005, pp. 1038–1044.Original Russian Text Copyright © 2005 by Prokop’ev, Sukhodolets.     

Recombinations occurring in the process of DNA replication in Escherichia coli

In a number of works dealing with the relationship between replication and recombination in bacteria, it is assumed that recombinations permit the replication forks to resume moving after having stopped at the damage sites of the template DNA. As an evidence for recombination occurring during DNA replication, the involvement in this process of proteins RuvABC and RecG, providing processing of the Holliday junctions after recombination, is considered. However, it has been shown that these proteins are not essential for resuming DNA synthesis after an exposure of bacteria to UV light. These data cast doubt on the necessity of recombination for reactivation of replication initiated in the oriC region. Studying recombination in tandem duplications in Escherichia coli showed that during replication, unequal crossing over occurs between direct DNA repeats of sister chromosomes. In wild strains, this crossing over results in tandem duplications, thereby enhancing the expression of certain genes. Thus, recombination of two types occurs during DNA replication: unequal crossing over leading to duplications and homologous exchange, responsible for post-replication DNA repair. The unequal exchange constitutes a component of SOS response of the cell to deterioration of the environment.     

Mechanisms for sister chromatid exchanges and their relation to the production of chromosomal aberrations

By taking advantage of the fact that fluorescent light (FL) induces strand breaks only in bromodeoxyuridine(BrdU)-substituted DNA, and that those breaks eventually lead to the formation of sister chromatid exchanges (SCEs), the response of SCEs to FL was studied carefully in Chinese hamster chromosomes in which, out of four DNA strands, BrdU-substitution had occurred either in one or three strands. The FL-induced SCE frequency did not differ greatly between these two types of chromosomes. However, when they were submitted to caffeine treatment, a drastic increase in the frequency was detected in the trifilarly-substituted chromosomes while a significant decrease occurred in the unifilarly-substituted chromosomes. Based on these results, a working hypothesis was developed that the SCE can arise by at least two different mechanisms, one operating at replicating points probably utilizing the machinery of DNA replication, and the other acting only in the post-replicational DNA portion, probably in a similar fashion as assumed in a general model of crossing over in the eukaryote. These dual mechanisms may account for the discrepancy encountered in the explanations of the induction of SCEs by various exogenous agents as well as spontaneous SCEs. The present study also showed that some, but clearly not all, of chromatid deletions are the outcome of the failure to complete SCEs arising through these mechanisms.     

Recombination and chromosome segregation

The duplication of DNA and faithful segregation of newly replicated chromosomes at cell division is frequently dependent on recombinational processes. The rebuilding of broken or stalled replication forks is universally dependent on homologous recombination proteins. In bacteria with circular chromosomes, crossing over by homologous recombination can generate dimeric chromosomes, which cannot be segregated to daughter cells unless they are converted to monomers before cell division by the conserved Xer site-specific recombination system. Dimer resolution also requires FtsK, a division septum-located protein, which coordinates chromosome segregation with cell division, and uses the energy of ATP hydrolysis to activate the dimer resolution reaction. FtsK can also translocate DNA, facilitate synapsis of sister chromosomes and minimize entanglement and catenation of newly replicated sister chromosomes. The visualization of the replication/recombination-associated proteins, RecQ and RarA, and specific genes within living Escherichia coli cells, reveals further aspects of the processes that link replication with recombination, chromosome segregation and cell division, and provides new insight into how these may be coordinated.     

Unequal crossing-over in Escherichia coli

Unequal crossing-over between sister chromosomes in the process of DNA replication in Escherichia coli leads to the formation of tandem duplications, thus enhancing the activity of certain genes. In conjugational matings between genetically marked E. coli strains, unequal crossing-over leads to the formation of heterozygous tandem duplications. Studying these duplications as model systems allowed the conclusion that unequal crossing-over between direct DNA repeats of sister chromosomes is the main pathway of the formation of selected recombinants in E. coli strains carrying duplications. This was inferred from the data on the segregation of homozygous diploid recombinants by heterozygous duplications. Unequal crossing-over between sister chromosomes occurs as adaptive exchange providing the survival of the greater part of bacterial cells on a selective medium. The known phenomenon of adaptive mutagenesis may also be a consequence of unequal exchanges at the level of DNA mononucleotide repeats.     

DNA-Replikation und Chromosomenstruktur von Mesostoma (Turbellaria)

During meiosis in M. ehrenbergi (2n=10) and M. lingua (2n=8) male certain chromosomes never pair completely. In these bivalents only terminal pairing appears, crossing over could not be proved by ³H-thymidine autoradiography. DNA amounts of the M. ehrenbergi and M. lingua genomes are in a proportion of 10∶1. The mitotic S-phase of spermatogonia in M. ehrenbergi is twice as long as in M. lingua. In metaphase of spermatogonia a differentiated DNA replication pattern can be identified in M. ehrenbergi as late-pulse-replicating segments. After incorporation of ³H-thymidine X₂-metaphase chromosomes can be found, which show single chromatid labeling, terminal and intercalary isolabeling as well as kinds of chromosome labeling, which can only result from sister strand exchange. After treating the chromosomes with low temperature, colchicine or by hydrolysis (60° C) substructures of the chromatin become visible in both spezies which however are evaluated as artefacts. — Formation of the different isolabeling types is discussed on the basis of a two-strand model of the chromosome fibril. A hypothesis is formulated that the surplusage of DNA in M. ehrenbergi is distributed over all the length of the chromatids as small parts of heterochromatin. This hypothesis is supported by investigations of the DNA replication and the contractility of the chromosomes. Furthermore, a pattern of small DNA particles can be demonstrated after partial destruction of the DNA in metaphase chromosomes of M. ehrenbergi, which could represent this intercalary heterochromatin.     

A matter of choice: the establishment of sister chromatid cohesion

Sister chromatid cohesion is the basis for the recognition of chromosomal DNA replication products for their bipolar segregation in mitosis. Fundamental to sister chromatid cohesion is the ring‐shaped cohesin complex, which is loaded onto chromosomes long before the initiation of DNA replication and is thought to hold replicated sister chromatids together by topological embrace. What happens to cohesin when the replication fork approaches, and how cohesin recognizes newly synthesized sister chromatids, is poorly understood. The characterization of a number of cohesion establishment factors has begun to provide hints as to the reactions involved. Cohesin is a member of the evolutionarily conserved family of Smc subunit‐based protein complexes that contribute to many aspects of chromosome biology by mediating long‐range DNA interactions. I propose that the establishment of cohesion equates to the selective stabilization of those cohesin‐mediated DNA interactions that link sister chromatids in the wake of replication forks.     

Studying recombination in heterozygous tandem duplications in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Our previous data showed that the principal pathway of the formation of selected recombinants in Escherichia coli strains carrying heterozygous tandem duplications is unequal crossing over between sister chromosomes. Data presented in this work showed that when DNA homology is not disturbed (due to transposon insertion), intragenic recombinants can occur directly in the region of recombination through intrachromomal exchange as well.     

Sister chromatid exchanges in the human active and inactive X chromosomes

Four human female fibroblast strains with an i(Xq) or derivative X chromosome as a cytological marker for the inactive X chromosome were used to determine the frequency of sister chromatid exchanges (SCEs) in the active and inactive X chromosomes. No significant difference in SCE frequency between the active and inactive X chromosomes was observed. Therefore, the state of chromatin condensation and the late DNA replication in the facultative heterochromatin of the inactive X chromosome do not appear to influence the SCE frequency.     

Replication-related activities establish cohesion between sister chromatids

Replicated sister chromatids are held together from their synthesis in S phase to their separation in anaphase. The process of sister chromatid cohesion is essential for the proper segregation of chromosomes in eukaroytic cells. Recent studies in Saccharomyces cerevisiae have advanced our understanding of how sister chromatid cohesion is established, maintained, and dissolved during the cell cycle. Historical observations have suggested that establishment of cohesion is roughly coincident with replication fork passage. Emerging evidence now indicates that replication fork components, such as PCNA, a novel DNA polymerase, Trf4p/Pol σ (formerly Trf4p/Pol κ), and a modified clamp-loader complex, actively participate in the process of the cohesion establishment. Here, we review the molecular events in the chromosome cycle with respect to cohesion. Failure of sister chromatid cohesion results in the aneuploidy characteristic of many birth defects and tumors in humans.     

Comparison of sister chromatid exchanges from three successive cell cycles in Wallabia bicolor chromosomes

An autoradiographic analysis of tritiated thymidine labeled chromosomes of Wallabia bicolor at the second and third metaphases after label incorporations has shown that sister chromatid exchanges (SCE's) from the first and second cell cycles are less than as frequent as SCE's from the third cell cycle after label. Exchange levels per cell cycle estimated at the seconf division are under-estimated due to coincident exchanges. In both methaphases exchanges were largely distributed at random along Wallabia chromosomes with frequencies proportional to chromosome length. The ratio of twin: single SCE's in spontaneoulsy occuring tetraploid cells indicated the first cycle exchanges were marginally more frequent than second cycle exchanges.These data are compatible with exchange probabilities being equal and independent over divisions, but a component of exchanges reducing as tritium content in chromosomes decreases cannot be excluded. This findings that SCE's are primarily independent of tritium cannot be attributed to a saturation of sites for exchange and it is therefore probable that sister exchanges are, in part at least, spontaneous events in Wallabia chromosomes.     

Automatic measurement of sister chromatid exchange frequency.

An automatic system for detecting and counting sister chromatid exchanges in human chromosomes has been developed. Metaphase chromosomes from lymphocytes which had incorporated 5-bromodeoxyuridine for two replication cycles were treated with the dye 33258 Hoechst and photodegraded so that the sister chromatids exhibited differential Giemsa staining. A computer-controlled television-microscope system was used to acquire digitized metaphase spread images by direct scanning of microscope slides. Individual objects in the images were identified by a thresholding procedure. The probability that each object was a single, separate chromosome was estimated from size and shape measurements. An analysis of the spatial relationships of the dark-chromatid regions of each object yielded a set of possible exchange locations and estimated probabilities that such locations corresponded to sister chromatid exchanges. A normalized estimate of the sister chromatid exchange frequency was obtained by summing the joint probabilities that a location contained an exchange within a single, separate chromosome over the set of chromosomes from one or more cells and dividing by the expected value of the total chromosome area analyzed. Comparison with manual scoring of exchanges showed satisfactory agreement up to levels of approximately 30 sister chromatid exchanges/cell, or slightly more than twice control levels. The processing time for this automated sister chromatid exchange detection system was comparable to that of manual scoring.     

On the continuity of chromosomal subunits: an analysis of induced ring chromosomes in Vicia faba

The proposition that subunits of a chromatid are continuous in a directional sense has been tested by observing the behaviour of induced ring chromosomes in Vicia faba. On the simplest hypothesis, that the subunits are the uninterrupted complementary strands of the DNA molecule, the polarity of rejoining should result in free separation of rings following replication in successive cell cycles. Centric and acentric ring chromosomes were separately assessed in both diploid and colchicine-accumulated tetraploid metaphase cells of primary root tips. Contrary to expectation large numbers of single and interlocked rings were observed in both cell cycles. Spontaneous sister chromatid exchanges and other breakage-reunion events can produce the configurations seen; with the postulated level of sister chromatid exchange equating that determined autoradiographically in rod chromosomes of V. faba. Unless the replication of ring chromosomes produces conditions unusual in rod chromosome replication, spontaneous breakage is probably common in replicating or post replication Vicia chromosomes. — A fundamental difference exists between the behaviour of centric and acentric ring chromosomes. Acentric ring chromosomes behave as if the chromatid arm were one DNA molecule, or a number of DNA molecules with identical directional sense. However, centric ring chromosomes behave as if there were a difference at the centromere in at least one (probably the metacentric) chromosome of the Vicia complement. That is, the two duplication-segregation subunits which extend the length of the chromosome, may contain a change in polarity at the centromere.     

Topoisomerase inhibitors can selectively interfere with different stages of simian virus 40 DNA replication.

I have found that antineoplastic drugs which are known to be inhibitors of mammalian DNA topoisomerases have pronounced and selective effects on simian virus 40 DNA replication. Ellipticine, 4'-(9-acridinylamino)methanesulfon-m-aniside, and Adriamycin blocked decatenation of newly replicated simian virus 40 daughter chromosomes in vivo. The arrested decatenation intermediates produced by these drugs contained single-strand DNA breaks. Ellipticine in particular produced these catenated dimers rapidly and efficiently. Removal of the drug resulted in rapid reversal of the block and completion of decatenation. The demonstration that these drugs interfere with decatenation suggests that they may exert their cytotoxic and antineoplastic effects by preventing the separation of newly replicated cellular chromosomes. Camptothecin rapidly breaks replication forks in growing Cairns structures. It is likely that the target of camptothecin is the "swivel" topoisomerase required for DNA replication and that it is located at or very near the replication fork in vivo. Evidence is presented that many of the broken Cairns structures are in fact half-completed sister chromatid exchanges. One pathway for the resolution of these structures is completion of the sister chromatid exchange to produce a circular head-to-tail dimer.     

Sister chromatid gene conversion is a prominent double-strand break repair pathway in mammalian cells

In mammalian cells, repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. By definition, homologous recombination requires a template with sufficient sequence identity to the damaged molecule in order to direct repair. We now show that the sister chromatid acts as a repair template in a substantial proportion of DSB repair events. The outcome of sister chromatid repair is primarily gene conversion unassociated with reciprocal exchange. This contrasts with expectations from the classical DSB repair model originally proposed for yeast meiotic recombination, but is consistent with models in which recombination is coupled intimately with replication. These results may explain why cytologically observable sister chromatid exchanges are induced only weakly by DNA-damaging agents that cause strand breaks, since most homologous repair events would not be observed. A preference for non-crossover events between sister chromatids suggests that crossovers, although genetically silent, may be disfavored for other reasons. Possibly, a general bias against crossing over in mitotic cells exists to reduce the potential for genome alterations when other homologous repair templates are utilized.