Replication dynamics in fission and budding yeasts through DNA polymerase tracking.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Tracking eukaryotic DNA polymerases during replication is challenging.
• Previous methods lacked resolution for studying replication dynamics.

Purpose of the Study:

• To develop novel methods for visualizing DNA polymerase dynamics in vivo.
• To investigate the organization of replicons and replication origins in yeast.
• To understand the causes of sequence instability during DNA replication.

Main Methods:

• Engineering replicative DNA polymerases to incorporate ribonucleotides at high rates.
• Utilizing engineered polymerases as tracers in Schizosaccharomyces pombe and Saccharomyces cerevisiae.
• Analyzing DNA polymorphisms and Okazaki fragment junctions on mononucleosomal DNA.

Main Results:

• Provided high-resolution insights into replicon and replication origin organization in two yeast species.
• Uncovered significant differences in replication dynamics between Schizosaccharomyces pombe and Saccharomyces cerevisiae.
• Identified sequence instability due to premature release of polymerase δ and retention of polymerase α-replicated DNA.

Conclusions:

• Engineered ribonucleotide-incorporating polymerases serve as effective tracers for DNA replication dynamics.
• This approach reveals distinct replication strategies between yeast species.
• Understanding polymerase dynamics and associated sequence instability is crucial for genomic stability, with potential applications in multicellular organisms.