Effizienter Support der DNA Replikationsfunktionen durch DNA Ligase 3 in Vertebraten

Die Ergebnisse in dieser Arbeit zeigen eine bemerkenswerte und bisher unerwartete Flexibilität in der DNA Replikation zwischen DNA Ligase 1 (LIG1) und DNA Ligase 3 (LIG3) in Vertebraten. Während die Letalität des LIG3 Knock-outs ausschließlich in der mitochondrialen Funktion und nicht in einer essentiellen Funktion im Zellnukleus begründet liegt, wird eindeutig gezeigt, dass LIG3 die Funktion von LIG1 in der Replikation voll übernehmen kann und dass DNA Ligase 4 (LIG4) nicht dazu in der Lage ist. Die Ergebnisse dieser Arbeit zeigen eindeutig, dass die Zink-Finger-Domäne von LIG3 essentiell für die Funktion von LIG3 in der Replikation ist, obwohl sie nicht essentiell für das Aufrechterhalten der Integrität der Mitochondrien ist. Außerdem wird gezeigt, dass die BRCT Domäne, mit deren Hilfe LIG3 mit XRCC1 interagiert, ebenfalls essentiell für die Rolle von LIG3 in der Replikation ist. In Co-Lokalisations-Experimenten konnte gezeigt werden, dass LIG3 zu Replikationszentren rekrutiert wird, wenn die Aktivität von LIG1 beeinträchtigt ist und dass die Rekrutierung von PCNA in diesem Hintergrund schwächer ist. Die Ergebnisse weisen auf einen unterschiedlichen Rekrutierungsmechanismus von LIG3 zu Replikationszentren hin im Vergleich zu dem für die Rekrutierung von LIG1 benötigten. Auch konnten die Mutanten, die in dieser Arbeit verwendet wurden, weitergehend charakterisiert werden in Bezug auf ihre Fähigkeit, Zellzyklus Kontrollpunkte nach Induktion von DNA Schäden zu aktivieren. Apoptose verkompliziert die Analyse des S-Phase Kontrollpunkts, da für eine verlässliche Quantifizierung hohe Dosen Strahlung benötigen werden. Andererseits zeigen DT40 Zellen einen normalen G2 Kontrollpunkt, der unbeeinträchtigt von Ligase Knock-outs bleibt.The results show a remarkable and hitherto unexpected functional flexibility in DNA replication of LIG1 and LIG3 in vertebrates. While the lethality of the LIG3 knockout is solely due to the mitochondrial function and not due to an essential function in the nucleus, it was conclusively shown that LIG3 can backup replication in the absence of LIG1 and that LIG4 is not capable to do so. The results in this thesis conclusively show that the ZnFn domain of LIG3 is essential for the DNA replication function of LIG3, although it is not essential for maintaining mitochondrial integrity. Furthermore, the BRCT domain with which LIG3 interacts with XRCC1 is also essential for the replication function of LIG3. In colocalization experiments it could be shown that LIG3 is recruited to DNA replication sites when LIG1 activity is impaired, and that the recruitment of PCNA is weakened. The results, therefore, point to a different recruitment system of LIG3 to replication factories compared to the one required for LIG1 recruitment. Furthermore the mutants used were characterized with regard to their ability to activate checkpoint responses after induction of DNA damage. Apoptosis complicates analysis of the S-phase checkpoint which requires high doses of radiation for reliable quantification. On the other hand, DT40 cells display a normal G2 checkpoint that remains unaffected by ligase deletion

Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells

In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excision (BER) and single strand break repair (SSBR). Here, using conditional knockout strategies for LIG3 and concomitant inactivation of the LIG1 and LIG4 genes, we show that in DT40 cells LigIII efficiently supports semi-conservative DNA replication. Our observations demonstrate a high functional versatility for the evolutionary new LigIII in DNA replication and mitochondrial metabolism, and suggest the presence of an alternative pathway for Okazaki fragment ligation

Stroke: Working Toward a Prioritized World Agenda

The aim of the Synergium was to devise and prioritize new ways of accelerating progress in reducing the risks, effects, and consequences of stroke

Stroke: Working Toward a Prioritized World Agenda

The aim of the Synergium was to devise and prioritize new ways of accelerating progress in reducing the risks, effects, and consequences of stroke

DNA ligases I and III cooperate in alternative non-homologous end-joining in vertebrates.

Biochemical and genetic studies suggest that vertebrates remove double-strand breaks (DSBs) from their genomes predominantly by two non-homologous end joining (NHEJ) pathways. While canonical NHEJ depends on the well characterized activities of DNA-dependent protein kinase (DNA-PK) and LIG4/XRCC4/XLF complexes, the activities and the mechanisms of the alternative, backup NHEJ are less well characterized. Notably, the contribution of LIG1 to alternative NHEJ remains conjectural and although biochemical, cytogenetic and genetic experiments implicate LIG3, this contribution has not been formally demonstrated. Here, we take advantage of the powerful genetics of the DT40 chicken B-cell system to delineate the roles of LIG1 and LIG3 in alternative NHEJ. Our results expand the functions of LIG1 to alternative NHEJ and demonstrate a remarkable ability for LIG3 to backup DSB repair by NHEJ in addition to its essential function in the mitochondria. Together with results on DNA replication, these observations uncover a remarkable and previously unappreciated functional flexibility and interchangeability between LIG1 and LIG3

LIG3 supports processing of DSBs also after low doses of radiation.

<p>(<b>A</b>) Representative images of γ-H2AX foci formation in wt, <i>LIG1^−/−</i>, <i>LIG4^−/−</i> and <i>LIG1^−/−LIG4^−/−</i> DT40 cells after exposure to 1 Gy X-rays at the indicated times after IR. (<b>B</b>) Representative kinetics of γ-H2A.X foci formation and decay of wt DT40 cells as measured by immunostaining after exposure to 0.5 and 1 Gy X-rays. The results shown represent the analysis of 4000 nuclei in one representative experiment. (<b>C</b>) γ-H2A.X foci scored in wt, <i>LIG1^−/−</i>, <i>LIG4^−/−</i> and <i>LIG1^−/−LIG4^−/−</i> cells 8 h after exposure to 1 Gy X-rays. Foci measured in non-irradiated cells have been subtracted. Results of two independent experiments, in which 8000 nuclei were scored, were used to calculate the indicated means and standard errors.</p

Dominant contribution of LIG3 <i>in-vitro</i> end joining.

<p>(<b>A</b>) Representative gels of <i>in vitro</i> DNA end joining of <i>Sal</i> I linearized <i>pSP65</i> plasmid using whole cell extracts of asynchronous wt, <i>LIG1^−/−</i>, <i>LIG4^−/−</i>, and <i>LIG1^−/−LIG4^−/−</i> cells. The linearized input substrate (linear) and the rejoined products (circles, dimers and multimers) generated by end joining are indicated (<b>B</b>) As in A. for <i>LIG3^−/2loxP</i> cells after treatment with 4HT for the indicated periods of time. (<b>C</b>) As in A. for <i>LIG3^−/2loxPLIG4</i>^−/− cells after treatment with 4HT for the indicated periods of time.</p

LIG3 processes IR-induced DSBs in LIG1 and LIG4 deficient cells.

<p>(<b>A</b>) Representative dose response curves for the induction of DSBs, as measured by PFGE, in cells exposed to increasing doses of X-rays. Images of ethidium bromide stained gels (upper panel) were analyzed to estimate the fraction of DNA released (FDR) from the well into the lane (regions defined as indicated) that is plotted as a function of IR dose for the indicated mutants (lower panel). Results from three independent experiments with 3 samples each were used to calculate the indicated means and standard errors. The dotted lines indicate the approach used to deduce Deq from FDR in DSB repair experiments (see text for details). (<b>B</b>) Repair kinetics of IR-induced DSBs in asynchronous wt, <i>LIG1^−/−</i>, <i>LIG4^−/−</i>, and <i>LIG1^−/−LIG4^−/−</i> cells after exposure to 40 Gy X-rays. The upper panel shows typical gels used to calculate the FDR at each repair time point, which was subsequently converted to Deq with the help of dose response curves such as those shown in A but generated with the same cell population used in the repair experiment (see text for details). Results of three determinations from at least two independent experiments were used to calculate the indicated means and standard errors (lower panel).</p

LIG1 and LIG3 contribute to the survival of cells exposed to IR.

<p>(<b>A</b>) Cell survival measured by colony formation in wt, <i>LIG1^−/−</i>, <i>LIG4</i>^−/− and <i>LIG1^−/−LIG4^−/−</i> cells after exposure to increasing doses of X-rays. Results from three independent experiments with 3 replicates each were used to calculate the indicated means and standard errors. (<b>B</b>) As in A. for <i>LIG3^−/2loxP</i>, and <i>LIG3^−/−Cdc9</i> cells after treatment for 1 h before and 4 h after IR with 10 µM NU7441. The dashed lines trace for comparison the results of wt and <i>LIG1^−/−LIG4</i>^−/− cells. (<b>C</b>) As in B. for <i>LIG3^−/2loxP</i>, <i>LIG3^−/M2I, LIG3^−/2loxPLIG4^−/−</i>, and <i>LIG3^−/M2ILIG4</i>^−/− cells. Results from at least two independent experiments with 3 replicates each were used to calculate the indicated means and standard errors. (<b>D</b>) As in B. for clone 3 of <i>LIG3^−/2loxPLIG4^−/−mts-hLIG1</i> and <i>LIG3^−/−LIG4^−/−mts-hLIG1.</i> Results from at least two independent experiments with 3 replicates each were used to calculate the indicated means and standard errors.</p

Conditional knockout of LIG3 reveals the function of LIG1 in the processing of IR-induced DSBs.

<p>(<b>A</b>) Kinetics of DSB processing in the indicated mutants after treatment with 4HT for the indicated periods of time. Other details are as in Fig. 1B. Results from three independent experiments with 3 samples each were used to calculate the indicated means and standard errors. (<b>B</b>) <i>LIG3</i> mRNA levels measured by real-time PCR in wt and <i>LIG3^−/2loxP</i> cells after different incubation times with 4HT. The mRNA level measured in wt cells was set to 100%. (<b>C</b>) Western blot analysis of LIG3 protein in <i>LIG3^−/2loxP</i> cells after treatment with 4HT for the indicated periods of time. A mouse monoclonal antibody against human LIG3 (clone 1F3) that recognizes the chicken LIG3 was used. GAPDH is a loading control. (<b>D</b>) Apoptotic index measured by microscopically scoring nuclear fragmentation and pycnosis in wt and <i>LIG3^−/2loxP</i> cells at various times after treatment with 4HT. Results from two independent experiments in each of which 1000 cells were scored were used to calculate the indicated means and standard errors. (<b>E</b>) As in A for the indicated mutants. (<b>F</b>) Representative cell-cycle distribution histograms obtained by flow cytometry in wt, <i>LIG4^−/−</i> and <i>LIG3^−/2loxPLIG4^−/−</i> cells treated with 4HT for 3.5 days before and after enrichment by centrifugal elutriation in G2 phase of the cell cycle. (<b>G</b>) Kinetics of DSB processing in cells enriched by centrifugal elutriation in the G2 phase of the cell cycle as shown in F.</p