Lotte Bjergbaek

DNA REPAIR. Mus81 and converging forks limit the mutagenicity of replication fork breakage.

How to repair broken replication forks Double-strand breaks in DNA are extremely dangerous to the integrity of our genomes. Most arise from problems encountered by replication forks during duplication of genomic DNA. Break-induced replication is known to use an error-prone DNA polymerase to repair such damage. Mayle et al. show that cells limit error-prone DNA synthesis by preventing the DNA polymerase from inadvertently switching to a related sequence with an incorrect template. The repair of the break is achieved by using a structure-specific nuclease to prevent formation of a long single-stranded region. Science, this issue p. 742 A nuclease prevents DNA polymerase from swinging to the wrong template while repairing broken DNA. Most spontaneous DNA double-strand breaks (DSBs) result from replication-fork breakage. Break-induced replication (BIR), a genome rearrangement–prone repair mechanism that requires the Pol32/POLD3 subunit of eukaryotic DNA Polδ, was proposed to repair broken forks, but how genome destabilization is avoided was unknown. We show that broken fork repair initially uses error-prone Pol32-dependent synthesis, but that mutagenic synthesis is limited to within a few kilobases from the break by Mus81 endonuclease and a converging fork. Mus81 suppresses template switches between both homologous sequences and diverged human Alu repetitive elements, highlighting its importance for stability of highly repetitive genomes. We propose that lack of a timely converging fork or Mus81 may propel genome instability observed in cancer.

RecQ helicases: lessons from model organisms

RecQ DNA helicases function during DNA replication and are essential for the maintenance of genome stability. There is increasing evidence that spontaneous genomic instability occurs primarily during DNA replication, and that proteins involved in the S-phase checkpoint are a principal defence against such instability. Cells that lack functional RecQ helicases exhibit phenotypes consistent with an inability to fully resume replication fork progress after encountering DNA damage or fork arrest. In this review we will concentrate on the various functions of RecQ helicases during S phase in model organisms.

DNA Topoisomerases Maintain Promoters in a State Competent for Transcriptional Activation in Saccharomyces cerevisiae

To investigate the role of DNA topoisomerases in transcription, we have studied global gene expression in Saccharomyces cerevisiae cells deficient for topoisomerases I and II and performed single-gene analyses to support our findings. The genome-wide studies show a general transcriptional down-regulation upon lack of the enzymes, which correlates with gene activity but not gene length. Furthermore, our data reveal a distinct subclass of genes with a strong requirement for topoisomerases. These genes are characterized by high transcriptional plasticity, chromatin regulation, TATA box presence, and enrichment of a nucleosome at a critical position in the promoter region, in line with a repressible/inducible mode of regulation. Single-gene studies with a range of genes belonging to this group demonstrate that topoisomerases play an important role during activation of these genes. Subsequent in-depth analysis of the inducible PHO5 gene reveals that topoisomerases are essential for binding of the Pho4p transcription factor to the PHO5 promoter, which is required for promoter nucleosome removal during activation. In contrast, topoisomerases are dispensable for constitutive transcription initiation and elongation of PHO5, as well as the nuclear entrance of Pho4p. Finally, we provide evidence that topoisomerases are required to maintain the PHO5 promoter in a superhelical state, which is competent for proper activation. In conclusion, our results reveal a hitherto unknown function of topoisomerases during transcriptional activation of genes with a repressible/inducible mode of regulation.

A Flp-nick system to study repair of a single protein-bound nick in vivo

We present the Flp-nick system, which allows introduction of a protein-bound nick at a single genomic site in Saccharomyces cerevisiae and thus mimics a stabilized topoisomerase I–DNA cleavage complex. We took advantage of a mutant Flp recombinase that can introduce a nick at a specific Flp recombinase recognition target site that has been integrated in the yeast genome. The genetic requirement for cells to cope with this insult is the same as for cells treated with camptothecin, which traps topoisomerase I–DNA cleavage complexes genome-wide. Hence, a single protein-bound nick is enough to kill cells if functional repair pathways are lacking. The Flp-nick system can be used to dissect repair, checkpoint and replication fork management pathways activated by a single genomic insult, and it allows the study of events at the damage site, which so far has been impossible to address.

Stimulated activity of human topoisomerases IIalpha and IIbeta on RNA-containing substrates.

Eukaryotic topoisomerase II is a dimeric nuclear enzyme essential for DNA metabolism and chromosome dynamics. Central to the activities of the enzyme is its ability to introduce transient double-stranded breaks in the DNA helix, where the two subunits of the enzyme become covalently attached to the generated 5′-ends through phosphotyrosine linkages. Here, we demonstrate that human topoisomerases IIα and IIβ are able to cleave ribonucleotide-containing substrates. With suicide substrates, which are partially double-stranded molecules containing a 5′-recessed strand, cleavage of both strands was stimulated ∼8-fold when a ribonucleotide rather than a deoxyribonucleotide was present at the scissile phosphodiester of the recessed strand. The existence of a ribonucleotide at the same position in a normal duplex substrate also enhanced topoisomerase II-mediated cleavage, although to a lesser extent. The enzyme covalently linked to the 5′-ribonucleotide in the cleavage complex efficiently performed ligation, and ligation occurred equally well to acceptor molecules terminated by either a 3′-ribo- or deoxyribonucleotide. Besides the enhanced topoisomerase II-mediated cleavage of ribonucleotide-containing substrates, cleavage of such substrates could be further stimulated by ATP or antitumor drugs. In conclusion, the observed in vitro activities of the human topoisomerase II isoforms indicate that the enzymes can operate on RNA or RNA-containing substrates and thus might possess an intrinsic RNA topoisomerase activity, as has previously been demonstrated forEscherichia coli topoisomerase III.

The Transducer Domain Is Important for Clamp Operation in Human DNA Topoisomerase IIα

DNA topoisomerase II is a multidomain homodimeric enzyme that changes DNA topology by coupling ATP hydrolysis to the transport of one DNA helix through a transient double-stranded break in another. The process requires dramatic conformational changes including closure of an ATP-operated clamp, which is comprised of two N-terminal domains from each protomer. The most N-terminal domain contains the ATP-binding site and is directly involved in clamp closure, undergoing dimerization upon ATP binding. The second domain, the transducer domain, forms the walls of the N-terminal clamp and connects the clamp to the enzyme core. Although structurally conserved, it is unclear whether the transducer domain is involved in clamp mechanism. We have purified and characterized a human topoisomerase IIα enzyme with a two-amino acid insertion at position 408 in the transducer domain. The enzyme retains both ATPase and DNA cleavage activities. However, the insertion, which is situated far from the N-terminal dimerization area, severely disrupts the function of the N-terminal clamp. The clamp-deficient enzyme is catalytically inactive and lacks most aspects of interdomain communication. Surprisingly, it seems to have retained the intersubunit communication, allowing it to bind ATP cooperatively in the presence of DNA. The results show that even distal parts of the transducer domain are important for the dynamics of the N-terminal clamp and furthermore indicate that stable clamp closure is not required for cooperative binding of ATP.

RecQ helicases and topoisomerase III in cancer and aging

RecQ helicases have in recent years attracted increasing attention due to the important roles they play in maintaining genomic integrity, which is essential for the life of a cell and the survival of a species. Humans with mutations in RecQ homologues are cancer prone and suffer from premature aging. A great effort has therefore been made to understand the molecular mechanisms and the biological pathways, in which RecQ helicases are involved. It has become clear that these enzymes work in close concert with DNA topoisomerase III, and studies in both yeast and mammalian systems point to a role of the proteins in processes involving homologous recombination. In this review we discuss the genetic and biochemical evidence for possible functions of RecQ helicases and DNA topoisomerase III in multiple cellular processes such as DNA recombination, DNA replication, and cell cycle checkpoint control.

Using a biochemical approach to identify the primary dimerization regions in human DNA topoisomerase IIalpha.

Eukaryotic topoisomerase II is a nuclear enzyme essential for DNA metabolism and chromosome dynamics. The enzyme has a dimeric structure, and subunit dimerization is vital to the cellular functions and activities of the enzyme. Two biochemical approaches based on metal ion affinity chromatography and immunoprecipitation have been carried out to map the dimerization region(s) in human topoisomerase IIα. The results demonstrate that two regions spanning amino acids 1053–1069 and 1124–1143 are both essential for dimerization. The regions correspond to the interaction domains revealed in yeast topoisomerase II after crystallization of a central fragment of this enzyme, indicating that the overall C-terminal dimerization structure of eukaryotic topoisomerase II is conserved from yeast to human. Furthermore, linker insertion analysis has demonstrated that the two dimerization regions are located in a highly flexible part of the enzyme. Topoisomerase IIα mutant enzymes unable to dimerize via the C-terminal primary dimerization regions due to lack of one of the defined dimerization regions can still be forced to dimerize if DNA and an ATP analog are added to the reaction mixture. The result indicates that secondary interactions occur by ATP analog-mediated clamp closing when the subunits are brought together on DNA.

MRX protects fork integrity at protein–DNA barriers, and its absence causes checkpoint activation dependent on chromatin context

To address how eukaryotic replication forks respond to fork stalling caused by strong non-covalent protein–DNA barriers, we engineered the controllable Fob-block system in Saccharomyces cerevisiae. This system allows us to strongly induce and control replication fork barriers (RFB) at their natural location within the rDNA. We discover a pivotal role for the MRX (Mre11, Rad50, Xrs2) complex for fork integrity at RFBs, which differs from its acknowledged function in double-strand break processing. Consequently, in the absence of the MRX complex, single-stranded DNA (ssDNA) accumulates at the rDNA. Based on this, we propose a model where the MRX complex specifically protects stalled forks at protein–DNA barriers, and its absence leads to processing resulting in ssDNA. To our surprise, this ssDNA does not trigger a checkpoint response. Intriguingly, however, placing RFBs ectopically on chromosome VI provokes a strong Rad53 checkpoint activation in the absence of Mre11. We demonstrate that proper checkpoint signalling within the rDNA is restored on deletion of SIR2. This suggests the surprising and novel concept that chromatin is an important player in checkpoint signalling.

A Rad53 independent function of Rad9 becomes crucial for genome maintenance in the absence of the Recq helicase Sgs1.

The conserved family of RecQ DNA helicases consists of caretaker tumour suppressors, that defend genome integrity by acting on several pathways of DNA repair that maintain genome stability. In budding yeast, Sgs1 is the sole RecQ helicase and it has been implicated in checkpoint responses, replisome stability and dissolution of double Holliday junctions during homologous recombination. In this study we investigate a possible genetic interaction between SGS1 and RAD9 in the cellular response to methyl methane sulphonate (MMS) induced damage and compare this with the genetic interaction between SGS1 and RAD24. The Rad9 protein, an adaptor for effector kinase activation, plays well-characterized roles in the DNA damage checkpoint response, whereas Rad24 is characterized as a sensor protein also in the DNA damage checkpoint response. Here we unveil novel insights into the cellular response to MMS-induced damage. Specifically, we show a strong synergistic functionality between SGS1 and RAD9 for recovery from MMS induced damage and for suppression of gross chromosomal rearrangements, which is not the case for SGS1 and RAD24. Intriguingly, it is a Rad53 independent function of Rad9, which becomes crucial for genome maintenance in the absence of Sgs1. Despite this, our dissection of the MMS checkpoint response reveals parallel, but unequal pathways for Rad53 activation and highlights significant differences between MMS- and hydroxyurea (HU)-induced checkpoint responses with relation to the requirement of the Sgs1 interacting partner Topoisomerase III (Top3). Thus, whereas earlier studies have documented a Top3-independent role of Sgs1 for an HU-induced checkpoint response, we show here that upon MMS treatment, Sgs1 and Top3 together define a minor but parallel pathway to that of Rad9.