+ Resolve Article
+ Follow Us
Follow on FacebookFollow on Facebook
Follow on TwitterFollow on Twitter

+ Translate
+ Subscribe to Site Feed
GeoScience Most Shared ContentMost Shared Content

Stimulation of RTH1 nuclease of the yeast Saccharomyces cerevisiae by replication protein A

, : Stimulation of RTH1 nuclease of the yeast Saccharomyces cerevisiae by replication protein A. Biochemistry 36(20): 5955-5962

The RTH1 nuclease is involved in the replication of chromosomal DNA as well as in the repair of DNA damage. Replication protein A (RPA) is also an integral part of the DNA replication and repair processes. We have investigated the roles(s) of RPA in the function of RTH1 nuclease, including its structure specific endonuclease activity. Initial in vitro studies, which employed a "flap" or a "pseudo Y" substrate containing a short 14 bp duplex region, showed the effect of RPA to be minimal or inhibitory. As RPA inhibition is unwarranted for a protein participating in the DNA replication process, we have further investigated the mechanism of such inhibition. Alternate flap and pseudo Y substrates with a long duplex region (50 bp) were prepared using M13mp19 ssDNA and synthetic oligonucleotides. Yeast RPA stimulated the endonuclease activity of RTH1 endonuclease with these substrates in a dose-dependent manner. Kinetic analysis suggested that yRPA exerted a bipartite effect on the nuclease reaction: (i) the "load time" of RTH1 nuclease onto the DNA substrate decreased from approximately 5 to 2 min in the presence of RPA, and (ii) following initiation of the nuclease reaction, the initial rate of the reaction increased 10-fold in the presence of yRPA. Further analysis of the interaction of RPA with various endonuclease substrates indicated that RPA has a weak helix destabilizing effect and could melt small, 14 bp, regions of duplex DNA. RTH1 endonuclease cleaves the DNA strand at the junction of single- and double-stranded DNA; consequently, the observed inhibition with small duplex substrates was likely due to duplex melting. Our studies also demonstrated that RPA stimulated the RNase H activity of RTH1 nuclease significantly. In both instances (RTH1 endonuclease and RNase H), the stimulation may involve a specific interaction of RPA with the RTH1 nuclease rather than a structural positioning of the DNA substrate by RPA.


PMID: 9166765

DOI: 10.1021/bi962890u

Other references

Al-Huthail, Y.R., 2006: Neuropsychiatric side-effects of interferon alfa therapy for hepatitis C and their management: a review. Interferon-Alfa (IFN-alpha) has been the mainstay of treatment for chronic hepatitis C. It has the potential to alter the course of chronic hepatitis C and prevent complications. This review summarizes current knowledge about the presentation, mec...

Lecuona, E.; Minin, A.; Trejo, H.E.; Chen, J.; Comellas, A.P.; Sun, H.; Grillo, D.; Nekrasova, O.E.; Welch, L.C.; Szleifer, I.; Gelfand, V.I.; Sznajder, J.I., 2010: Myosin-Va restrains the trafficking of Na+/K+-ATPase-containing vesicles in alveolar epithelial cells. Stimulation of Na(+)/K(+)-ATPase activity in alveolar epithelial cells by cAMP involves its recruitment from intracellular compartments to the plasma membrane. Here, we studied the role of the actin molecular motor myosin-V in this process. We pro...

Saunders G.C., 1982: Sensitive new method for determination of elastolytic activity using enzyme labeled elastin. Federation Proceedings 41(3): ABSTRACT 322

Li, X.; Yu, H-Ying., 2015: Characterization of an organic solvent-tolerant lipase from Haloarcula sp. G41 and its application for biodiesel production. A haloarchaeal strain G41 showing lipolytic activity was isolated from the saline soil of Yuncheng Salt Lake, China. Biochemical and physiological characterizations along with 16S rRNA gene sequence analysis placed the isolate in the genus Haloarc...

Hong, Felix, T., 1995: Fundamentals of photoelectric effects in molecular electronic thin film devices: Applications to bacteriorhodopsin-based devices. This tutorial lecture focuses on the fundamental mechanistic aspects of light-induced charge movements in pigment-containing membranes. The topic is relevant to molecular electronics because many prototypes optoelectronic devices are configured as...

Terzic Vidojevic, A., 1992: Microorganisms in raw milk. Microbial growth in raw milk is discussed with reference to: milk composition; sources of microbial contamination; types of contaminating microorganisms; and effects of time and temperature on microbial growth. Bacteria frequently found in milk (i...

Zhang, J.-J.; Liu, T.; Peng, F., 2012: Primary Paraganglioma of the Lung A Case Report and Literature Review. Only 22 cases of primary pulmonary paraganglioma have been reported previously. This report presents a case of paraganglioma that arose in the lung of a 38-year-old woman who presented with chest pain and paroxysmal cough with little sputum. Posit...

Belloni, B.; Andres, C., 2011: Images in clinical medicine: Extravasation of peripherally administered parenteral nutrition. New England Journal of Medicine 364(10): E20-E20

Khalil, M.K.; Nadrah, H.M.; Al-Yahia, O.A.; Al-Saigul, A.M., 2008: Sero-response to measles vaccination at 12 months of age in Saudi infants in Qassim Province. Saudi Medical Journal 29(7): 1009-1013

MacDermid, J.C., 2016: Sex and gender in hand therapy research. Journal of Hand Therapy 29(1): 1-2