Srs2 and Pif1 as Model Systems for Understanding Sf1a and Sf1b Helicase Structure and Function

Abstract

Helicases are enzymes that convert the chemical energy stored in ATP into mechanical work, allowing them to move along and manipulate nucleic acids. The helicase superfamily 1 (Sf1) is one of the largest subgroups of helicases and they are required for a range of cellular activities across all domains of life. Sf1 helicases can be further subdivided into two classes called the Sf1a and Sf1b helicases, which move in opposite directions on nucleic acids. The results of this movement can range from the separation of strands within duplex nucleic acids to the physical remodeling or removal of nucleoprotein complexes. Here, we describe the characteristics of the Sf1a helicase Srs2 and the Sf1b helicase Pif1, both from the model organism Saccharomyces cerevisiae, focusing on the roles that they play in homologous recombination, a DNA repair pathway that is necessary for maintaining genome integrity.

A four-point molecular handover during Okazaki maturation

“…2c), no density was observed for the endonuclease domain, indicating that it is flexible in this structure. Next, we used a version of Pol I that lacks the N-terminal endonuclease domain (also known as the Klenow fragment, Pol I KL ) to collect a large cryo-EM dataset of more than 11,000 images and more helicases 21,22 , where the fingers domain of Pol I acts like a strand separating pin, while the primer extension of Pol I drives DNA translocation, analogous to the ATPase domains of the monomeric helicases. We furthermore present the biochemical analysis of the sequential activities of the four enzymes involved in the Okazaki maturation.…”

The translocation activity of Rad54 reduces crossover outcomes during homologous recombination

“…The end point of the homology search is the extension of D-loops, which allows the transition to the DNA repair and resolution phase of HR (23). In Saccharomyces cerevisiae, the steps in the homology search are regulated by several DNA translocases and helicases, including Rad54 (24), Rdh54 (14,(25)(26)(27), and Srs2 (13,(28)(29)(30)(31)(32). These ATP-dependent motors control intermediates, including Rad51 filaments (28,29,32,33) and D-loop structures (13,33,34).…”

The separation pin distinguishes the pro– and anti–recombinogenic functions of Saccharomyces cerevisiae Srs2

“…The S. cerevisiae protein Srs2 is an Sf1a family member that has served as a paradigm for understanding helicase–mediated regulation of HR 6 , 35 , 36 . HR is an essential DNA repair pathway that allows for the exchange of genetic information between two different DNA molecules of identical or nearly identical sequence composition and is essential for the maintenance of genome integrity 37 – 39 .…”