A single-stranded DNA binding protein required for mitochondrial DNA replication in S. cerevisiae is homologous to E. coli SSB.

Dyck, Eric Van; Foury, Françoise; Stillman, Bruce; Brill, Steven J.

doi:10.1002/j.1460-2075.1992.tb05421.x

Cited by 170 publications

(121 citation statements)

References 51 publications

Supporting

Mentioning

114

Contrasting

Order By: Relevance

“…2. Among the proteins that were expected to be crosslinked to mtDNA, we identified Abf2p (4), Rim1p [mitochondrial ssDNA binding protein (20)], and Mgm101p (7,8). These proteins have been shown to bind mtDNA in vitro and in vivo by a combination of cosedimentation, fluorescence microscopy (green fluorescent protein-tagged proteins or indirect immunofluorescence), and direct DNA binding studies.…”

Section: Resultsmentioning

confidence: 99%

In organello formaldehyde crosslinking of proteins to mtDNA: Identification of bifunctional proteins

Kaufman

Newman

Hallberg

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

186

139

View full text Add to dashboard Cite

The segregating unit of mtDNA is a protein-DNA complex called the nucleoid. In an effort to understand how nucleoid proteins contribute to mtDNA organization and inheritance, we have developed an in organello formaldehyde crosslinking procedure to identify proteins associated with mtDNA. Using highly purified mitochondria, we observed a time-dependent crosslinking of protein to mtDNA as determined by sedimentation through isopycnic cesium chloride gradients. We detected Ϸ20 proteins crosslinked to mtDNA and identified 11, mostly by mass spectrometry. Among them is Abf2p, an abundant, high-mobility group protein that is known to function in nucleoid morphology, and in mtDNA transactions. In addition to several other proteins with known DNA binding properties or that function in mtDNA maintenance, we identified other mtDNA-associated proteins that were not anticipated, such as the molecular chaperone Hsp60p and a Krebs cycle protein, Kgd2p. Genetic experiments indicate that hsp60-ts mutants have a petite-inducing phenotype at the permissive temperature and that a kgd2⌬ mutation increases the petite-inducing phenotype of an abf2⌬ mutation. Crosslinking and DNA gel shift experiments show that Hsp60p binds to single-stranded DNA with high specificity for the template strand of a putative origin of mtDNA replication. These data identify bifunctional proteins that participate in the stability of ؉ mtDNA.

show abstract

Section: Resultsmentioning

confidence: 99%

In organello formaldehyde crosslinking of proteins to mtDNA: Identification of bifunctional proteins

Kaufman

Newman

Hallberg

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

186

139

View full text Add to dashboard Cite

show abstract

“…Several key enzymes involved in mtDNA maintenance are closely related to their prokaryotic counterparts; these include mitochondrial RNA polymerase (3,21), SSB protein (4,9,20,31), the high-mobility-group-box DNA binding protein mitochondrial transcription factor A (25), and the catalytic subunit of DNA pol ␥ (17,26,27,34). Nevertheless, the observation that the small subunit of DNA pol ␥ is related to prokaryotic aminoacyl-tRNA synthetases is surprising, since the latter represent a different class of enzyme involved in nucleic acid metabolism.…”

Section: Resultsmentioning

confidence: 99%

The Accessory Subunit of Xenopus laevis Mitochondrial DNA Polymerase γ Increases Processivity of the Catalytic Subunit of Human DNA Polymerase γ and Is Related to Class II Aminoacyl-tRNA Synthetases

Carrodeguas

Kobayashi

Lim

et al. 1999

Molecular and Cellular Biology

102

View full text Add to dashboard Cite

Peptide sequences obtained from the accessory subunit of Xenopus laevis mitochondrial DNA (mtDNA) polymerase ␥ (pol ␥) were used to clone the cDNA encoding this protein. Amino-terminal sequencing of the mitochondrial protein indicated the presence of a 44-amino-acid mitochondrial targeting sequence, leaving a predicted mature protein with 419 amino acids and a molecular mass of 47.3 kDa. This protein is associated with the larger, catalytic subunit in preparations of active mtDNA polymerase. The small subunit exhibits homology to its human, mouse, and Drosophila counterparts. Interestingly, significant homology to glycyl-tRNA synthetases from prokaryotic organisms reveals a likely evolutionary relationship. Since attempts to produce an enzymatically active recombinant catalytic subunit of Xenopus DNA pol ␥ have not been successful, we tested the effects of adding the small subunit of the Xenopus enzyme to the catalytic subunit of human DNA pol ␥ purified from baculovirus-infected insect cells. These experiments provide the first functional evidence that the small subunit of DNA pol ␥ stimulates processive DNA synthesis by the human catalytic subunit under physiological salt conditions. Mitochondrial DNA (mtDNA) is replicated by a DNA polymerase, DNA polymerase ␥ (pol ␥), that is distinct from nuclear DNA polymerases ␣, ␤, ␦, ε, and . Since DNA pol ␥ represents only a small fraction of total cellular DNA polymerase, purification and characterization of the subunit composition of this enzyme have been difficult. Molecular cloning has contributed greatly to understanding the structure of DNA pol ␥ in different organisms. The catalytic subunits of DNA pol ␥ have been cloned for several organisms (6,16,17,27,34) and have been found to resemble family A of DNA polymerases, related to Escherichia coli DNA pol I. In Saccharomyces cerevisiae DNA pol ␥ is composed of a single polypeptide, while in Drosophila melanogaster DNA pol ␥ is comprised of two different polypeptides, a catalytic subunit of 125 kDa and an accessory subunit of 41 kDa (24, 33). The Drosophila subunits copurify and have been shown to interact, but the recombinant proteins have not yet been shown to be functional. The function of the small subunit, which we refer to as pol ␥B, is unknown. It has been proposed to influence the processivity of the catalytic subunit. Putative mammalian homologs of the Drosophila accessory subunit have been identified in sequence databases. One published purification scheme for human DNA pol ␥ suggested the existence of a small subunit (11), but the potential relationship between this polypeptide and Drosophila pol ␥B has not been established. Recently, the catalytic subunit of human DNA pol ␥ was expressed in an active form (10,19). Surprisingly, the recombinant catalytic subunit alone displayed most of the characteristics of the enzyme purified from human cells, which did not appear to contain a stoichiometric amount of a small subunit. Thus, it is not clear what role, if any, is played by putative human DNA pol ␥B.We hav...

show abstract

“…This suggests that the core histones are present in a large complex or several subcomplexes (which always contain H2A, H2B, H3, and H4). Among these were proteins of the replication factor A (RPA) complex, which is involved in DNA replication, but also proteins involved in DNA recombination and repair (reviewed in (39,40)): the Yku complex, which also functions in nonhomologous end joining (NHEJ), DNA repair (34) and Rim1, the homolog of SSB (singlestranded DNA binding protein) in E. coli (41), and subunits of RNA polymerase III.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Highly Conserved Histone Related Protein, Ydl156w, and Its Functional Associations Using Quantitative Proteomic Analyses

Gilmore

Sardiu

Venkatesh

et al. 2012

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

A significant challenge in biology is to functionally annotate novel and uncharacterized proteins. Several approaches are available for deducing the function of proteins in silico based upon sequence homology and physical or genetic interaction, yet this approach is limited to proteins with well-characterized domains, paralogs and/or orthologs in other species, as well as on the availability of suitable large-scale data sets. Here, we present a quantitative proteomics approach extending the protein network of core histones H2A, H2B, H3, and H4 in Saccharomyces cerevisiae, among which a novel associated protein, the previously uncharacterized Ydl156w, was identified. In order to predict the role of Ydl156w, we designed and applied integrative bioinformatics, quantitative proteomics and biochemistry approaches aiming to infer its function. Reciprocal analysis of Ydl156w protein interactions demonstrated a strong association with all four histones and also to proteins strongly associated with histones including Rim1, Rfa2 and 3, Yku70, and Yku80. Through a subsequent combination of the focused quantitative proteomics experiments with available largescale genetic interaction data and Gene Ontology functional associations, we provided sufficient evidence to associate Ydl156w with multiple processes including chromatin remodeling, transcription and DNA repair/replication. To gain deeper insights into the role of Ydl156w in histone biology we investigated the effect of the genetic deletion of ydl156w on H4 associated proteins, which lead to a dramatic decrease in the association of H4 with RNA polymerase III proteins. The implication of a role for Ydl156w in RNA Polymerase III mediated transcription was consequently verified by RNA-Seq experiments. Finally, using these approaches we generated a refined network of Ydl156w-associated proteins. Molecular &

show abstract

A single-stranded DNA binding protein required for mitochondrial DNA replication in S. cerevisiae is homologous to E. coli SSB.

Cited by 170 publications

References 51 publications

In organello formaldehyde crosslinking of proteins to mtDNA: Identification of bifunctional proteins

In organello formaldehyde crosslinking of proteins to mtDNA: Identification of bifunctional proteins

The Accessory Subunit of Xenopus laevis Mitochondrial DNA Polymerase γ Increases Processivity of the Catalytic Subunit of Human DNA Polymerase γ and Is Related to Class II Aminoacyl-tRNA Synthetases

Characterization of a Highly Conserved Histone Related Protein, Ydl156w, and Its Functional Associations Using Quantitative Proteomic Analyses

Contact Info

Product

Resources

About