scholarly journals Canonical and novel non-canonical activities of the Holliday junction resolvase Yen1

2020 ◽  
Author(s):  
F. Javier Aguado ◽  
Raquel Carreira ◽  
Vanesa Hurtado-Nieves ◽  
Miguel G. Blanco
Keyword(s):  
Budding Yeast ◽  
Holliday Junction ◽  
Conformational Preference ◽  
Alternative Processing ◽  
Holliday Junction Resolvase ◽  
Replication Intermediates

ABSTRACTYen1 and GEN1 are members of the Rad2/XPG family of nucleases that were identified as the first canonical nuclear Holliday junction (HJ) resolvases in budding yeast and humans due to their ability to introduce two symmetric, coordinated incisions on opposite strands of the HJ, yielding nicked DNA products that could be readily ligated. While GEN1 has been extensively characterized in vitro, much less is known about the biochemistry of Yen1. Here, we have performed the first in-depth characterization of purified Yen1. We confirmed that Yen1 resembles GEN1 in many aspects, including range of substrates targeted, position of most incisions they produce or monomeric state in solution. However, we have also observed unexpected alternative processing of substrates, such as nicked HJs and a different conformational preference on intact HJs. Moreover, we demonstrate that Yen1 is endowed with additional nuclease activities, like a nick-specific 5’-3’ exonuclease or HJ arm-chopping that could apparently blur its classification as a canonical HJ resolvase. Despite this, we show that Yen1 fulfills the requirements of a canonical HJ resolvase and hypothesize that its wider array of nuclease activities might contribute to its function in the removal of persistent recombination or replication intermediates.

scholarly journals Isolation and characterization of a gene (CBF2) specifying a protein component of the budding yeast kinetochore.

1993 ◽  
Vol 121 (3) ◽  
pp. 513-519 ◽  
Author(s):  
W Jiang ◽  
J Lechner ◽  
J Carbon
Keyword(s):  
Molecular Motors ◽  
Cell Biology ◽  
Budding Yeast ◽  
Isolation And Characterization ◽  
Sequence Homologies ◽  
Binding Factor ◽  
Microtubule Motor

We have cloned and determined the nucleotide sequence of the gene (CBF2) specifying the large (110 kD) subunit of the 240-kD multisubunit yeast centromere binding factor CBF3, which binds selectively in vitro to yeast centromere DNA and contains a minus end-directed microtubule motor activity. The deduced amino acid sequence of CBF2p shows no sequence homologies with known molecular motors, although a consensus nucleotide binding site is present. The CBF2 gene is essential for viability of yeast and is identical to NDC10, in which a conditional mutation leads to a defect in chromosome segregation (Goh, P.-Y., and J. V. Kilmartin, in this issue of The Journal of Cell Biology). The combined in vitro and in vivo evidence indicate that CBF2p is a key component of the budding yeast kinetochore.

scholarly journals Quantitative Characterization of a Mitotic Cyclin Threshold Regulating Exit from Mitosis

2005 ◽  
Vol 16 (5) ◽  
pp. 2129-2138 ◽  
Author(s):  
Frederick R. Cross ◽  
Lea Schroeder ◽  
Martin Kruse ◽  
Katherine C. Chen
Keyword(s):  
Cell Cycle ◽  
Budding Yeast ◽  
Cell Cycle Control ◽  
Predictive Ability ◽  
Eukaryotic Cell ◽  
Model Predictions ◽  
Mitotic Cyclin ◽  
Constitutive Overexpression

Regulation of cyclin abundance is central to eukaryotic cell cycle control. Strong overexpression of mitotic cyclins is known to lock the system in mitosis, but the quantitative behavior of the control system as this threshold is approached has only been characterized in the in vitro Xenopus extract system. Here, we quantitate the threshold for mitotic block in budding yeast caused by constitutive overexpression of the mitotic cyclin Clb2. Near this threshold, the system displays marked loss of robustness, in that loss or even heterozygosity for some regulators becomes deleterious or lethal, even though complete loss of these regulators is tolerated at normal cyclin expression levels. Recently, we presented a quantitative kinetic model of the budding yeast cell cycle. Here, we use this model to generate biochemical predictions for Clb2 levels, asynchronous as well as through the cell cycle, as the Clb2 overexpression threshold is approached. The model predictions compare well with biochemical data, even though no data of this type were available during model generation. The loss of robustness of the Clb2 overexpressing system is also predicted by the model. These results provide strong confirmation of the model's predictive ability.

scholarly journals Sequence Specificity and Biochemical Characterization of the RusA Holliday Junction Resolvase ofEscherichia coli

1997 ◽  
Vol 272 (23) ◽  
pp. 14873-14882 ◽  
Author(s):  
Sau N. Chan ◽  
Lynda Harris ◽  
Edward L. Bolt ◽  
Matthew C. Whitby ◽  
Robert G. Lloyd
Keyword(s):  
Biochemical Characterization ◽  
Ofescherichia Coli ◽  
Holliday Junction ◽  
Sequence Specificity ◽  
Holliday Junction Resolvase

scholarly journals Replication intermediates that escape Dna2 activity are processed by Holliday junction resolvase Yen1

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Gizem Ölmezer ◽  
Maryna Levikova ◽  
Dominique Klein ◽  
Benoît Falquet ◽  
Gabriele Alessandro Fontana ◽  
...  
Keyword(s):  
Holliday Junction ◽  
Holliday Junction Resolvase ◽  
Replication Intermediates

Localization and regulation of the cdk-activating kinase (Cak1p) from budding yeast

1998 ◽  
Vol 111 (24) ◽  
pp. 3585-3596 ◽  
Author(s):  
P. Kaldis ◽  
Z.W. Pitluk ◽  
I.A. Bany ◽  
D.A. Enke ◽  
M. Wagner ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Budding Yeast ◽  
Subcellular Fractionation ◽  
Eukaryotic Cell ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cell Cycles ◽  
Cdk Activating Kinase

Eukaryotic cell cycles are controlled by the activities of cyclin-dependent kinases (cdks). The major cdk in budding yeast, Saccharomyces cerevisiae, is Cdc28p. Activation of Cdc28p requires phosphorylation on threonine 169 and binding to a cyclin. Thr-169 is phosphorylated by the cdk-activating kinase (CAK), Cak1p, which was recently identified as the physiological CAK in budding yeast. Here we present our further characterization of yeast Cak1p. We have found that Cak1p is dispersed throughout the cell as shown by immunofluorescence; biochemical subcellular fractionation confirmed that most of the Cak1p is found in the cytoplasm. Cak1p is a monomeric enzyme in crude yeast lysates. Mutagenesis of potential sites of activating phosphorylation had little effect on the activity of Cak1p in vitro or in vivo. Furthermore, Cak1p contains no posttranslational modifications detectable by two-dimensional isoelectric focusing. We found that Cak1p is a stable protein during exponential growth but that its expression decreases considerably when cells enter stationary phase. In contrast, Cak1p levels oscillate dramatically during meiosis, reflecting regulation at both the transcriptional and post-translational level. The localization and regulation of Cak1p are in contrast to those of the known vertebrate CAK, p40(MO15).

Sign in / Sign up

Export Citation Format

Share Document