Regulation der Genomstabilität durch SUMO- und Ubiquitin-Modifikation von PCNA

Eine akkurate DNA-Replikation ist notwendig, um die Stabilität der genetischen Information zu gewährleisten. Dieser Prozess wird durch DNA-Läsionen erschwert, die durch eine Vielzahl von Ursachen entstehen und häufig nicht vor dem Erreichen der S-Phase repariert werden können. Nicht nur kann durch Läsionen geschädigte DNA häufig nicht dupliziert werden, angehaltene Replikationsgabeln können auch zusammenbrechen und so zu DNA-Strangbrüchen führen. Die Funktion des RAD6-pathways liegt darin, die Umgehung (Bypass) von DNA-Läsionen während der Replikation zu ermöglichen, wodurch eine Toleranz gegenüber Schädigungen der DNA erreicht wird. In dieser Arbeit wurde die Regulation des RAD6-vermittelten Bypass von DNA-Läsionen durch posttranslationale Ubiquitin- und SUMO-Modifikationen des Replikationsfaktors PCNA untersucht. PCNA bildet einen trimeren Ring um die DNA und verstärkt durch Bindung der replikativen Polymerase deren Assoziation zur DNA und somit die Prozessivität der Replikation. Als DNA gebundener Faktor des Replikations-komplexes ohne katalytische Aktivität ist PCNA ideal geeignet, um durch seine Modifikation Replikations-assoziierte Prozesse zu regulieren. Die Ubiquitinierung von PCNA durch Enzyme des RAD6-pathways erfolgt als spezifische Antwort auf DNA-Läsionen während der Replikation und ermöglicht deren Bypass. Dabei bewirken unterschiedliche Ubiquitin-Modifikationen verschiedene Arten des Bypass. Die Mono-Ubiquitin-Modifikation führt zum Einsatz von speziellen Transläsions-Polymerasen, die eine größere Toleranz für geschädigte DNA haben, aber auch für die Entstehung von Mutationen verantwortlich sind. Einen mechanistisch anderen Bypass von DNA-Schäden bewirkt die Modifikation von PCNA mit einer Lysin K63-verknüpften Multi-Ubiquitinkette. Für diesen wird wahrscheinlich der neureplizierte, unbeschädigte Schwester-Strang als Vorlage benutzt. Unabhängig von Schädigungen der DNA wird PCNA während der S-Phase zusätzlich mit dem ubiquitin-ähnlichen Protein SUMO modifiziert. Dies führt zu einer Interaktion mit der Helikase Srs2, die als Antagonist zu dem zentralen Rekombinationsprotein Rad51 wirkt. Dadurch wird spezfisch die homologe Rekombination zwischen Schwesterchromatiden an der Rekombinationsgabel inhibiert, nicht jedoch andere Rekombinationsereignisse, wie. z.B. Rekom-bination zwischen homologen Chromosomen. Deshalb ist es wahrscheinlich, dass spezifisch die Replikationsgabel durch PCNA-SUMO-Srs2 geschützt wird, um schädliche Rekombination oder Rekombinationsstrukturen zu vermeiden, die mit Replikations-assoziierten Prozessen interferieren. Ubiquitin- und SUMO-Modifikation regulieren demnach unabhängige Prozesse. Interessanterweise haben diese aber eine verwandte Funktion im Bypass von DNA-Läsionen während der Replikation. Die Inhibition der Schwesterchromatid-Rekombination durch PCNA-SUMO-Srs2 lenkt den Bypass von DNA-Läsionen in einen durch PCNA-Ubiquitinierung gesteuerten Mechanismus

NUCLEOSOMES DYNAMICS AT DNA DOUBLE STRAND BREAKS

Background: DNA double-strand breaks (DSBs) are highly toxic lesions that, if not correctly repaired, can have detrimental consequences on genome integrity and cell survival. Repair of DSBs by homologous recombination (HR) requires the processing of DSB ends during DNA end resection, which, through the degradation of 5’-terminated strands, generates a long stretch of single-stranded DNA (ssDNA). Therefore, resection divides the chromatin surrounding a DSB in distinct ssDNA and dsDNA domains. The molecular composition of these domains is crucial for HR repair as well as for DNA damage signaling and checkpoint activation. However, it was unclear whether nucleosomes, the fundamental unit of chromatin, could be found in the ssDNA domain as well. [...

Nucleolar release of rDNA repeats for repair involves SUMO-mediated untethering by the Cdc48/p97 segregase

Ribosomal RNA genes (rDNA) are highly unstable and susceptible to rearrangement due to their repetitive nature and active transcriptional status. Sequestration of rDNA in the nucleolus suppresses uncontrolled recombination. However, broken repeats must be first released to the nucleoplasm to allow repair by homologous recombination. Nucleolar release of broken rDNA repeats is conserved from yeast to humans, but the underlying molecular mechanisms are currently unknown. Here we show that DNA damage induces phosphorylation of the CLIP-cohibin complex, releasing membrane-tethered rDNA from the nucleolus in Saccharomyces cerevisiae. Downstream of phosphorylation, SUMOylation of CLIP-cohibin is recognized by Ufd1 via its SUMO-interacting motif, which targets the complex for disassembly through the Cdc48/p97 chaperone. Consistent with a conserved mechanism, UFD1L depletion in human cells impairs rDNA release. The dynamic and regulated assembly and disassembly of the rDNA-tethering complex is therefore a key determinant of nucleolar rDNA release and genome integrity

Unscheduled DNA replication in G1 causes genome instability and damage signatures indicative of replication collisions

DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineer genetic systems in budding yeast to induce unscheduled replication in a G1- like cell cycle state. Unscheduled G1 replication initiates at canonical S-phase origins. We quantifiy the composition of replisomes in G1- and S-phase and identified firing factors, polymerase α, and histone supply as factors that limit replication outside S-phase. G1 replication per se does not trigger cellular checkpoints. Subsequent replication during S-phase, however, results in overreplication and leads to chromosome breaks and chromosome-wide, strandbiased occurrence of RPA-bound single-stranded DNA, indicating head-to-tail replication collisions as a key mechanism generating genome instability upon G1 replication. Low-level, sporadic induction of G1 replication induces an identical response, indicating findings from synthetic systems are applicable to naturally occurring scenarios of unscheduled replication initiation

Human Holliday junction resolvase GEN1 uses a chromodomain for efficient DNA recognition and cleavage

Abstract Holliday junctions (HJs) are key DNA intermediates in homologous recombination. They link homologous DNA strands and have to be faithfully removed for proper DNA segregation and genome integrity. Here, we present the crystal structure of human HJ resolvase GEN1 complexed with DNA at 3.0 Å resolution. The GEN1 core is similar to other Rad2/XPG nucleases. However, unlike other members of the superfamily, GEN1 contains a chromodomain as an additional DNA interaction site. Chromodomains are known for their chromatin-targeting function in chromatin remodelers and histone(de)acetylases but they have not previously been found in nucleases. The GEN1 chromodomain directly contacts DNA and its truncation severely hampers GEN1's catalytic activity. Structure-guided mutations in vitro and in vivo in yeast validated our mechanistic findings. Our study provides the missing structure in the Rad2/XPG family and insights how a well-conserved nuclease core acquires versatility in recognizing diverse substrates for DNA repair and maintenance

A CDK-regulated chromatin segregase promoting chromosome replication

The replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood. Here, we report the characterization of a chromatin remodeling enzyme-Yta7-entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+ -ATPase that assembles into \~1 MDa hexameric complexes capable of segregating histones from DNA. The Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that the enzymatic activity of Yta7 is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication. Our results present a mechanism for how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase

Structural mechanism of extranucleosomal DNA readout by the INO80 complex

The nucleosomal landscape of chromatin depends on the concerted action of chromatin remodelers. The INO80 remodeler specifically places nucleosomes at the boundary of gene regulatory elements, which is proposed to be the result of an ATP-dependent nucleosome sliding activity that is regulated by extranucleosomal DNA features. Here, we use cryo–electron microscopy and functional assays to reveal how INO80 binds and is regulated by extranucleosomal DNA. Structures of the regulatory A-module bound to DNA clarify the mechanism of linker DNA binding. The A-module is connected to the motor unit via an HSA/post-HSA lever element to chemomechanically couple the motor and linker DNA sensing. Two notable sites of curved DNA recognition by coordinated action of the four actin/actin-related proteins and the motor suggest how sliding by INO80 can be regulated by extranucleosomal DNA features. Last, the structures clarify the recruitment of YY1/Ies4 subunits and reveal deep architectural similarities between the regulatory modules of INO80 and SWI/SNF complexes

Dpb11 coordinates Mec1 kinase activation with cell cycle-regulated Rad9 recruitment

Cyclin-dependent kinase phosphorylation of the replication checkpoint mediator Rad9 controls its association with Dpb11, a key activator of the yeast ATR homologue Mec1, thus conferring cell-cycle dependence to checkpoint signalling

MRE11 complex links RECQ5 helicase to sites of DNA damage

RECQ5 DNA helicase suppresses homologous recombination (HR) possibly through disruption of RAD51 filaments. Here, we show that RECQ5 is constitutively associated with the MRE11–RAD50–NBS1 (MRN) complex, a primary sensor of DNA double-strand breaks (DSBs) that promotes DSB repair and regulates DNA damage signaling via activation of the ATM kinase. Experiments with purified proteins indicated that RECQ5 interacts with the MRN complex through both MRE11 and NBS1. Functional assays revealed that RECQ5 specifically inhibited the 3′→5′ exonuclease activity of MRE11, while MRN had no effect on the helicase activity of RECQ5. At the cellular level, we observed that the MRN complex was required for the recruitment of RECQ5 to sites of DNA damage. Accumulation of RECQ5 at DSBs was neither dependent on MDC1 that mediates binding of MRN to DSB-flanking chromatin nor on CtIP that acts in conjunction with MRN to promote resection of DSBs for repair by HR. Collectively, these data suggest that the MRN complex recruits RECQ5 to sites of DNA damage to regulate DNA repair