| 1. |
- Lara-Gonzalez, Samuel, et al.
(författare)
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Substrate-induced dimerization of engineered monomeric variants of triosephosphate isomerase from trichomonas vaginalis
- 2015
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 Å2. TIMs from Trichomonas vaginalis (TvTIM) are held in their dimeric state by two mechanisms: A ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A) is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.
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| 2. |
- Trasviña-Arenas, Carlos H., et al.
(författare)
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Amino and carboxy-terminal extensions of yeast mitochondrial DNA polymerase assemble both the polymerization and exonuclease active sites
- 2019
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Ingår i: Mitochondrion. - : Elsevier BV. - 1567-7249 .- 1872-8278. ; 49, s. 166-177
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Tidskriftsartikel (refereegranskat)abstract
- Human and yeast mitochondrial DNA polymerases (DNAPs), POLG and Mip1, are related by evolution to bacteriophage DNAPs. However, mitochondrial DNAPs contain unique amino and carboxyl-terminal extensions that physically interact. Here we describe that N-terminal deletions in Mip1 polymerases abolish polymerization and decrease exonucleolytic degradation, whereas moderate C-terminal deletions reduce polymerization. Similarly, to the N-terminal deletions, an extended C-terminal deletion of 298 amino acids is deficient in nucleotide addition and exonucleolytic degradation of double and single-stranded DNA. The latter observation suggests that the physical interaction between the amino and carboxyl-terminal regions of Mip1 may be related to the spread of pathogenic POLG mutant along its primary sequence.
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| 3. |
- Sanchez Sandoval, Maria Eugenia, 1985, et al.
(författare)
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Yeast mitochondrial RNA polymerase primes mitochondrial DNA polymerase at origins of replication and promoter sequences
- 2015
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Ingår i: Mitochondrion. - : Elsevier BV. - 1567-7249 .- 1872-8278. ; 24, s. 22-31
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Tidskriftsartikel (refereegranskat)abstract
- Three proteins phylogenetically grouped with proteins from the T7 replisome localize to yeast mitochondria: DNA polymerase γ (Mip1), mitochondrial RNA polymerase (Rpo41), and a single-stranded binding protein (Rim1). Human and T7 bacteriophage RNA polymerases synthesize primers for their corresponding DNA polymerases. In contrast, DNA replication in yeast mitochondria is explained by two models: a transcription-dependent model in which Rpo41 primes Mip1 and a model in which double stranded breaks create free 3' OHs that are extended by Mip1. Herein we found that Rpo41 transcribes RNAs that can be extended by Mip1 on single and double-stranded DNA. In contrast to human mitochondrial RNA polymerase, which primes DNA polymerase γ using transcripts from the light-strand and heavy-strand origins of replication, Rpo41 primes Mip1 at replication origins and promoter sequences in vitro. Our results suggest that in ori1, short transcripts serve as primers, whereas in ori5 an RNA transcript longer than 29 nucleotides is used as primer.
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| 4. |
- Trasvinã-Arenas, Carlos H., et al.
(författare)
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Dispensability of the [4Fe-4S] cluster in novel homologues of adenine glycosylase MutY
- 2016
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Ingår i: FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 283, s. 521-540
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Tidskriftsartikel (refereegranskat)abstract
- 7,8-Dihydro-8-deoxyguanine (8oG) is one of the most common oxidative lesions in DNA. DNA polymerases misincorporate an adenine across from this lesion. Thus, 8oG is a highly mutagenic lesion responsible for G:C→T:A transversions. MutY is an adenine glycosylase, part of the base excision repair pathway that removes adenines, when mispaired with 8oG or guanine. Its catalytic domain includes a [4Fe-4S] cluster motif coordinated by cysteinyl ligands. When this cluster is absent, MutY activity is depleted and several studies concluded that the [4Fe-4S] cluster motif is an indispensable component for DNA binding, substrate recognition and enzymatic activity. In the present study, we identified 46 MutY homologues that lack the canonical cysteinyl ligands, suggesting an absence of the [4Fe-4S] cluster. A phylogenetic analysis groups these novel MutYs into two different clades. One clade is exclusive of the order Lactobacillales and another clade has a mixed composition of anaerobic and microaerophilic bacteria and species from the protozoan genus Entamoeba. Structural modeling and sequence analysis suggests that the loss of the [4Fe-4S] cluster is compensated by a convergent solution in which bulky amino acids substitute the [4Fe-4S] cluster. We functionally characterized MutYs from Lactobacillus brevis and Entamoeba histolytica as representative members from each clade and found that both enzymes are active adenine glycosylases. Furthermore, chimeric glycosylases, in which the [4Fe-4S] cluster of Escherichia coli MutY is replaced by the corresponding amino acids of LbY and EhY, are also active. Our data indicates that the [4Fe-4S] cluster plays a structural role in MutYs and evidences the existence of alternative functional solutions in nature.
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