DNA ligase 4 stabilizes the ribosomal DNA array upon fork collapse at the replication fork barrier

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• DNA double-strand breaks (DSBs) at replication fork barriers in ribosomal DNA (rDNA) of Saccharomyces cerevisiae are poorly understood.
• Previous studies using 2D-gel electrophoresis indicated DSBs but lacked detailed characterization of their origin, nature, and magnitude.

Purpose of the Study:

• To quantify DSBs at the rDNA replication fork barrier in wild-type and mutant yeast cells.
• To elucidate the origin and repair mechanisms of these DSBs.
• To investigate the role of Dnl4 ligase in rDNA stability and DSB processing.

Main Methods:

• Quantification of DSBs using 2D-gel electrophoresis in wild-type and sgs1 mutant Saccharomyces cerevisiae.
• Analysis of DSB detectability in the presence and absence of functional Dnl4 ligase.
• Assessment of extrachromosomal ribosomal DNA circle formation.

Main Results:

• Approximately 14% of replicating rDNA molecules exhibit breaks at the replication fork barrier in wild-type cells, equating to 7-10 DSBs per cell during S-phase.
• Breaks observed in wild-type cells are likely artifacts from nicks near the replication fork barrier (rRFB), not true DSBs.
• A distinct class of DSBs, dependent on functional Dnl4, is identified in sgs1 mutant cells with compromised replication fork stability.
• Dnl4 activity limits both the formation of these DSBs and extrachromosomal rDNA circles.
• dnl4 mutant cells show altered rDNA replication fork structures.

Conclusions:

• The study identifies a novel class of DNA double-strand breaks specifically in sgs1 mutant yeast cells, highlighting compromised replication fork stability.
• Functional Dnl4 ligase plays a critical role in preventing these DSBs and limiting extrachromosomal rDNA circle formation, thereby maintaining rDNA stability.
• Dnl4 has an unrecognized function in maintaining the integrity of the ribosomal DNA locus during replication.