The inter-conversion of telomeres between different functional states, defined by the composition of the bound proteins, is the fundamental step that dictates whether the chromosome end is recognized as a bona-fide end or incorrectly as a double-strand break. In recent years it has become evident that motor proteins (e.g. helicases) play a role in telomere regulation. Even in yeast, one of the best-studied model systems, how helicases function in telomere regulation is not fully understood. For example, it is not clear whether the major function of these motor proteins originate from their helicase activity and thus their ability to unwind double-stranded nucleic acid or from their ability to translocate along DNA and remove bound proteins. In S.cerevisiae, Pif1 helicase affects telomere function via displacement of the telomerase, the specialized reverse transcriptase responsible for extension of the G-rich strand of the telomere. A second helicase, Rrm3, has been implicated in telomere function. Rrm3 facilitates replication fork progression at >1000 chromosomal sites, including telomeres. A possible function of Rrm3 in replication fork progression at telomeres is the displacement of Rap1 bound to the duplex region of the telomere. Knowledge of the intrinsic biochemical properties of Pif1 and Rrm3 and how they are employed/altered to carry out their functions is necessary to elucidate how these motor proteins participate in telomere regulation.
In Specific Aim 1 we will: A) Study the allosteric modulation by nucleotide cofactors of Pif1-DNA interactions. B) Determine which oligomeric state of Pif1 is responsible for its activity as a helicase or a translocase and how Pif1 catalyzes these activities. C) Test whether Pif1 inhibition of the telomerase originates from its helicase activity or its ability to displace subunits of the telomerase complex.
In Specific Aim 2 we will: A) Test our hypothesis that the ATPase and helicase activities of Rrm3 are regulated by a sequence/domain within its N-terminal region. B) Test the hypothesis that PCNA binding to Rrm3 relieves the regulatory function of its N-terminal region. C) Test the hypothesis that interaction with PCNA leads to an active Rrm3 that is able to displace Rap1 from the telomere. These studies will allow us to determine how Pif1 and Rrm3 function and will provide unique insight on how these enzymes participate in telomere/telomerase regulation. We expect to define whether oligomerization of Pif1 leads to separation of its functions and thus to its specific role at telomeres. Also, we expect to determine how the activities of Rrm3 can be regulated to allow removal of proteins bound to double-stranded region of the telomere, thus facilitating replication of these sites.

Public Health Relevance

The proposed research is relevant to public health because chromosome instability, oncogenesis and cellular senescence are linked to the de-regulation of telomerase function and telomere integrity. The activity of helicases plays an important level of regulation of both the telomerase and the organization of the telomere. The proper recognition of the end of chromosomes as a true end leads to chromosome maintenance otherwise it is incorrectly processed as a double-strand break. Our knowledge of the role of motor protein activity at telomeres is of fundamental importance for our understanding of their function as regulators. Thus, the proposed research focuses on the study of their mechanism and it is relevant to the NIH's mission to develop fundamental knowledge that will help understand human diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM098509-05
Application #
8897389
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Carter, Anthony D
Project Start
2011-08-01
Project End
2017-07-31
Budget Start
2015-08-01
Budget End
2017-07-31
Support Year
5
Fiscal Year
2015
Total Cost
Indirect Cost
Name
Washington University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Singh, Saurabh P; Kukshal, Vandna; De Bona, Paolo et al. (2018) The mitochondrial single-stranded DNA binding protein from S. cerevisiae, Rim1, does not form stable homo-tetramers and binds DNA as a dimer of dimers. Nucleic Acids Res 46:7193-7205
Dahan, Danielle; Tsirkas, Ioannis; Dovrat, Daniel et al. (2018) Pif1 is essential for efficient replisome progression through lagging strand G-quadruplex DNA secondary structures. Nucleic Acids Res 46:11847-11857
Geronimo, Carly L; Singh, Saurabh P; Galletto, Roberto et al. (2018) The signature motif of the Saccharomyces cerevisiae Pif1 DNA helicase is essential in vivo for mitochondrial and nuclear functions and in vitro for ATPase activity. Nucleic Acids Res 46:8357-8370
Koc, Katrina N; Singh, Saurabh P; Stodola, Joseph L et al. (2016) Pif1 removes a Rap1-dependent barrier to the strand displacement activity of DNA polymerase ?. Nucleic Acids Res 44:3811-9
Singh, Saurabh P; Koc, Katrina N; Stodola, Joseph L et al. (2016) A Monomer of Pif1 Unwinds Double-Stranded DNA and It Is Regulated by the Nature of the Non-Translocating Strand at the 3'-End. J Mol Biol 428:1053-1067
Sokoloski, Joshua E; Kozlov, Alexander G; Galletto, Roberto et al. (2016) Chemo-mechanical pushing of proteins along single-stranded DNA. Proc Natl Acad Sci U S A 113:6194-9
Feldmann, Erik A; De Bona, Paolo; Galletto, Roberto (2015) The wrapping loop and Rap1 C-terminal (RCT) domain of yeast Rap1 modulate access to different DNA binding modes. J Biol Chem 290:11455-66
Koc, Katrina N; Stodola, Joseph L; Burgers, Peter M et al. (2015) Regulation of yeast DNA polymerase ?-mediated strand displacement synthesis by 5'-flaps. Nucleic Acids Res 43:4179-90
Feldmann, Erik A; Koc, Katrina N; Galletto, Roberto (2015) Alternative arrangements of telomeric recognition sites regulate the binding mode of the DNA-binding domain of yeast Rap1. Biophys Chem 198:1-8
Feldmann, Erik A; Galletto, Roberto (2014) The DNA-binding domain of yeast Rap1 interacts with double-stranded DNA in multiple binding modes. Biochemistry 53:7471-83

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