The Yeast Mitochondrial RNA Polymerase and Transcription Factor Complex Catalyzes Efficient Priming of DNA Synthesis on Single-stranded DNA.
Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast mitochondrial DNA polymerase Mip1. However, the ssDNA-binding protein Rim1 severely inhibits the RNA synthesis activity of Rpo41, but not the Rpo41-Mtf1 complex, which continues to prime DNA synthesis efficiently in the presence of Rim1. We show that RNAs as short as 10-12 nt serve as primers for DNA synthesis. Characterization of the RNA-DNA products shows that Rpo41 and Rpo41-Mtf1 have slightly different priming specificity. However, both prefer to initiate with ATP from short priming sequences such as 3'-TCC, TTC, and TTT, and the consensus sequence is 3'-Pu(Py)2-3 Based on our studies, we propose that Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA.
From the Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
Aparna
A
Ramachandran
From the Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, Graduate School of Biomedical Sciences, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
Divya
D
Nandakumar
From the Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
Aishwarya P
AP
Deshpande
Department of Pharmacology and Toxicology, Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, Texas 77555, and.
Thomas P
TP
Lucas
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205.
Ramanagouda
R
R-Bhojappa
From the Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
Guo-Qing
GQ
Tang
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205.
Kevin
K
Raney
Department of Pharmacology and Toxicology, Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, Texas 77555, and.
Y Whitney
YW
Yin
From the Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, patelss@rutgers.edu.
Smita S
SS
Patel
D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000096:biosynthesis* / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004271:DNA, Fungal; D004277:DNA, Single-Stranded; C071720:MTF1 protein, S cerevisiae; D024101:Mitochondrial Proteins; D046912:Multiprotein Complexes; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases; C487996:RPO41 protein, S cerevisiae; D004256:DNA Polymerase I; C068352:MIP1 protein, S cerevisiae
DNA primase; DNA replication; RNA polymerase; mitochondrial DNA (mtDNA); transcription factor