Structural analysis of DNA replication across unstable repetitive sequences

Die  Verkürzung  oder  Expansion  von  sich  wiederholenden  Trinukleotidesequenzen,  sogenannten  „Trinukleotid‐repeats“  (TNR),  ist  die  Ursache  für  neurodegenerative  Krankheiten  wie  Friedreichs  Ataxie  (GAA),  die  Huntington‐Krankheit  (CAG)  oder  das  Fragile‐X‐Syndrom  (CGG).  Lange  TNR  Sequenzen  können  alternative  DNS‐ Sekundärstrukturen  in  vitro  bilden  und  hemmen  das  Fortschreiten  von  DNS  Replikationsgabeln  in  Hefezellen  und  Bakterien.  In  menschlichen  Zellen  sind  die molekularen Mechanismen, die die DNS Replikation beeinträchtigen und zur Expansion  der  TNR  führen,  allerdings  weitgehend  unbekannt.  Wir  haben  ein  experimentelles  System etabliert, um die in vivo Replikationsstrukturen („replication intermediates“, RI)  zu analysieren, die bei der DNS Replikation von GAA‐Trinukleotidsequenzen entstehen.  Dabei  transfizieren  wir  humane  Zellen  mit  Plasmiden,  die  GAA‐Sequenzen  in  unterschiedlichen  Längen  und  Orientierungen  enthalten.  Nach  Replikation  dieser  Plasmide  in  den  transfizierten  Zellen  isolieren  wir  die  RI  und  analysieren  sie  mittels  bidimensionalen (2D) Agarosegeln und dem Elektronenmikroskop (EM).  Unsere  2D‐Gel‐Analysen  von  RI  aus  humanen  293T  und  U2OS  Zellen  zeigt,  dass  Replikationsgabeln  durch  GAA‐Sequenzen  nur  transient  angehalten  werden,  und  dass  dieser  Effekt  von  der  Länge  und  Orientierung  der  TNR  abhängt.  Zu  unserer  Überraschung haben wir ausserdem noch weitere Signale in unseren 2D‐Gelen erhalten,  deren  Auftreten  mit  der  Länge  von  TNR,  bei  der  Symptome  von  Friedreichs  Ataxie  (FRDA)  auftreten,  korreliert.  Mit  Hilfe  des  EM  haben  wir  sowohl  die  gesamte  RI  Population,  als  auch  die  Moleküle,  die  wir  durch  Elution  der  genannten  Signale  aus  unseren 2D‐Gelen isoliert haben, umfassend analysiert. Dabei haben wir erstmals hoch  aufgelöste  Bilder  der  Strukturen  gewonnen,  mit  denen  Schwesterchromatiden  unmittelbar  hinter  der  Replikationsgabel  miteinander  verbunden  sind.  Bei  ungestörter  Replikation  sind  diese  Verbindungen  willkürlich  über  die  gesamte  Länge  der  replizierten Moleküle verteilt. Im Gegensatz dazu führen expandierte GAA‐Sequenzen zu  einer  Stabilisierung  dieser  Verbindungen  in  der  repetitiven  Sequenz.  Darüber  hinaus  führen  GAA‐Sequenzen  zur  Reversion  der  Replikationsgabel  in  vivo  und  beeinflussen  gleichzeitig  die  Stabilität  der  zweiten  Replikationsgabel  des  Replikons.  Die  Ergebnisse  unsere  Experimente  legen  nahe,  dass  postreplikative  Strukturen  für  die  GAA Triplettexpansion  und  damit  für  das  Auftreten  von  Friedreichs  Ataxie  verantwortlich  sind.  Ähnliche  Vorgänge  könnten  ursächlich  für  die  Expansion  andere  TNR‐Sequenzen  sein,  die  mit  einer  wachsenden  Zahl  neurodegenerativer  Erkrankungen  in  Verbindung  gebracht werden.  Die  experimentelle  Identifikation  an  der  Expansion  von  GAA‐Sequenzen  beteiligter  zellulärer Faktoren und die Entwicklung effektiver Diagnosetechniken sind bisher durch  methodische  Schwierigkeiten  bei  der  Detektion  expandierter  TNR  eingeschränkt.  Für  eine  effektive  Diagnose  und  ein  tieferes  Verständnis  der  molekularen  Grundlagen  der  FRDA  sind  die  schnelle  und  zuverlässige  Detektion  expandierter  TNR  aber  Voraussetzung.  Ausgehend  von  isolierter  DNS  mit  GAA‐Sequenzen  und  den  damit  verbundenen alternativen Strukturen haben wir in Zusammenarbeit mit der Gruppe von  Dr. Toshio Mori einen Antikörper etabliert, der spezifisch DNS Epitope in expandierten  GAA‐Sequenzen  erkennt.  Unsere  in  vitro  Experimente  haben  die  Spezifität  dieses  Antikörpers  bestätigt,  aber  auch  gezeigt,  dass  eine  Detektion  von  GAA‐assoziierten  Strukturen  in  vivo  aufgrund  des  hohen  Überschusses  normaler  DNS  mit  diesem  Antikörper nicht möglich ist. Daher haben wir uns auf die Verfeinerung unserer in vitro  Techniken  konzentriert,  um  das  analytische  Potential  dieses  Antikörpers  optimal  auszunutzen  und  zusätzliche  Informationen  über  den  Einfluss  von  GAA‐Sequenzen  sowohl auf die DNS Replikation als auch auf die Transkription zu gewinnen

BRCA1 controls homologous recombination at Tus/Ter-stalled mammalian replication forks

Replication fork stalling can promote genomic instability, predisposing to cancer and other diseases1–3. Stalled replication forks may be processed by sister chromatid recombination (SCR), generating error-free or error-prone homologous recombination (HR) outcomes4–8. In mammalian cells, a long-standing hypothesis proposes that the major hereditary breast/ovarian cancer predisposition gene products, BRCA1 and BRCA2, control HR/SCR at stalled replication forks9. Although BRCA1 and BRCA2 affect replication fork processing10–12, direct evidence that BRCA genes regulate HR at stalled chromosomal replication forks is lacking due to a dearth of tools for studying this process. We report that the Escherichia coli Tus/Ter complex13–16 can be engineered to induce site-specific replication fork stalling and chromosomal HR/SCR in mammalian cells. Tus/Ter-induced HR entails processing of bidirectionally arrested forks. We find that the BRCA1 C-terminal tandem BRCT repeat and regions of BRCA1 encoded by exon 11—two BRCA1 elements implicated in tumor suppression—control Tus/Ter-induced HR. Inactivation of either BRCA1 or BRCA2 increases the absolute frequency of “long-tract” gene conversions at Tus/Ter-stalled forks—an outcome not observed in response to a restriction endonuclease-mediated chromosomal double strand break (DSB). Therefore, HR at stalled forks is regulated differently from HR at DSBs arising independently of a fork. We propose that aberrant long-tract HR at stalled replication forks contributes to genomic instability and breast/ovarian cancer predisposition in BRCA mutant cells

Combined bidimensional electrophoresis and electron microscopy to study specific plasmid DNA replication intermediates in human cells

Replication interference by specific chromosomal sequences—such as trinucleotide repeats—plays a causative, though undefined role in the aetiology of human disease, especially neurodegenerative syndromes. However, studies on these mechanisms in human cells have been hampered by poorly defined replication origins on genomic DNA. Simian Virus 40 (SV40)-based plasmids were useful in the past to overcome these experimental limits, but have been rarely amenable for the most complex and revealing molecular biology approaches to study in vivo DNA replication interference. This chapter describes a new, safe, SV40-based episomal system that replicates with very high efficiency in human cells and allows isolation of in vivo replication intermediates with high yield and purity. We describe how to use this experimental system to run preparative agarose 2D-gel and to extract specific replication intermediates to visualize by electron microscopy

Rif1 Binding and Control of Chromosome-Internal DNA Replication Origins Is Limited by Telomere Sequestration

The Saccharomyces cerevisiae telomere-binding protein Rif1 plays an evolutionarily conserved role in control of DNA replication timing by promoting PP1-dependent dephosphorylation of replication initiation factors. However, ScRif1 binding outside of telomeres has never been detected, and it has thus been unclear whether Rif1 acts directly on the replication origins that it controls. Here, we show that, in unperturbed yeast cells, Rif1 primarily regulates late-replicating origins within 100 kb of a telomere. Using the chromatin endogenous cleavage ChEC-seq technique, we robustly detect Rif1 at late-replicating origins that we show are targets of its inhibitory action. Interestingly, abrogation of Rif1 telomere association by mutation of its Rap1-binding module increases Rif1 binding and origin inhibition elsewhere in the genome. Our results indicate that Rif1 inhibits replication initiation by interacting directly with origins and suggest that Rap1-dependent sequestration of Rif1 increases its effective concentration near telomeres, while limiting its action at chromosome-internal sites

Exo1 competes with repair synthesis, converts NER intermediates to long ssDNA gaps, and promotes checkpoint activation

Ultraviolet (UV) light induces DNA-damage checkpoints and mutagenesis, which are involved in cancer protection and tumorigenesis, respectively. How cells identify DNA lesions and convert them to checkpoint-activating structures is a major question. We show that during repair of UV lesions in noncycling cells, Exo1-mediated processing of nucleotide excision repair (NER) intermediates competes with repair DNA synthesis. Impediments of the refilling reaction allow Exo1 to generate extended ssDNA gaps, detectable by electron microscopy, which drive Mec1 kinase activation and will be refilled by long-patch repair synthesis, as shown by DNA combing. We provide evidence that this mechanism may be stimulated by closely opposing UV lesions, represents a strategy to redirect problematic repair intermediates to alternative repair pathways, and may also be extended to physically different DNA damages. Our work has significant implications for understanding the coordination between repair of DNA lesions and checkpoint pathways to preserve genome stability

Noncanonical Mismatch Repair as a Source of Genomic Instability in Human Cells

Mismatch repair (MMR) is a key antimutagenic process that increases the fidelity of DNA replication and recombination. Yet genetic experiments showed that MMR is required for antibody maturation, a process during which the immunoglobulin loci of antigen-stimulated B cells undergo extensive mutagenesis and rearrangements. In an attempt to elucidate the mechanism underlying the latter events, we set out to search for conditions that compromise MMR fidelity. Here, we describe noncanonical MMR (ncMMR), a process in which the MMR pathway is activated by various DNA lesions rather than by mispairs. ncMMR is largely independent of DNA replication, lacks strand directionality, triggers PCNA monoubiquitylation, and promotes recruitment of the error-prone polymerase-η to chromatin. Importantly, ncMMR is not limited to B cells but occurs also in other cell types. Moreover, it contributes to mutagenesis induced by alkylating agents. Activation of ncMMR may therefore play a role in genomic instability and cancer

Visualization of recombination-mediated damage bypass by template switching

Template switching (TS) mediates damage bypass via a recombination-related mechanism involving PCNA polyubiquitination and polymerase δ-dependent DNA synthesis. Using two-dimensional gel electrophoresis and EM, here we characterize TS intermediates arising in Saccharomyces cerevisiae at a defined chromosome locus, identifying five major families of intermediates. Single-stranded DNA gaps of 150-200 nt, and not DNA ends, initiate TS by strand invasion. This causes reannealing of the parental strands and exposure of the nondamaged newly synthesized chromatid, which serves as a replication template for the other blocked nascent strand. Structures resembling double Holliday junctions, postulated to be central double-strand break-repair intermediates but so far visualized only in meiosis, mediate late stages of TS before being processed to hemicatenanes. Our results reveal the DNA transitions accounting for recombination-mediated DNA-damage tolerance in mitotic cells and replication under conditions of genotoxic stress

Friedreich's ataxia-associated GAA repeats induce replication-fork reversal and unusual molecular junctions

Expansion of GAA/TTC repeats is the causative event in Friedreich's ataxia. GAA repeats have been shown to hinder replication in model systems, but the mechanisms of replication interference and expansion in human cells remained elusive. To study in vivo replication structures at GAA repeats, we designed a new plasmid-based system that permits the analysis of human replication intermediates by two-dimensional gel electrophoresis and EM. We found that replication forks transiently pause and reverse at long GAA/TTC tracts in both orientations. Furthermore, we identified replication-associated intramolecular junctions, located between GAA/TTC repeats and other homopurine-homopyrimidine tracts, that were associated with breakage of the plasmid fork not traversing the repeats. Finally, we detected postreplicative, sister-chromatid hemicatenanes on control plasmids, which were replaced by persistent homology-driven junctions at GAA/TTC repeats. These data prove that GAA/TTC tracts interfere with replication in humans and implicate postreplicative mechanisms in trinucleotide repeat expansion