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1.  Ddc2 Mediates Mec1 Activation through a Ddc1- or Dpb11-Independent Mechanism 
PLoS Genetics  2014;10(2):e1004136.
The protein kinase Mec1 (ATR ortholog) and its partner Ddc2 (ATRIP ortholog) play a key role in DNA damage checkpoint responses in budding yeast. Previous studies have established the model in which Ddc1, a subunit of the checkpoint clamp, and Dpb11, related to TopBP1, activate Mec1 directly and control DNA damage checkpoint responses at G1 and G2/M. In this study, we show that Ddc2 contributes to Mec1 activation through a Ddc1- or Dpb11-independent mechanism. The catalytic activity of Mec1 increases after DNA damage in a Ddc2-dependent manner. In contrast, Mec1 activation occurs even in the absence of Ddc1 and Dpb11 function at G2/M. Ddc2 recruits Mec1 to sites of DNA damage. To dissect the role of Ddc2 in Mec1 activation, we isolated and characterized a separation-of-function mutation in DDC2, called ddc2-S4. The ddc2-S4 mutation does not affect Mec1 recruitment but diminishes Mec1 activation. Mec1 phosphorylates histone H2A in response to DNA damage. The ddc2-S4 mutation decreases phosphorylation of histone H2A more significantly than the absence of Ddc1 and Dpb11 function does. Our results suggest that Ddc2 plays a critical role in Mec1 activation as well as Mec1 localization at sites of DNA damage.
Author Summary
When DNA replication is blocked and DNA damage occurs, checkpoints arrest the cell cycle in eukaryotic cells, allowing DNA replication and repair to take place. The major regulators of the DNA damage checkpoint response are the phosphoinositide 3-kinase (PI3K)-related protein kinases, including ATM and ATR. In budding yeast, ATM and ATR correspond to Tel1 and Mec1, respectively. ATM/Tel1 acts in response to double-strand breaks. By contrast, ATR/Mec1 recognizes many different types of DNA damage. Mec1 forms a complex with Ddc2 (ATRIP ortholog) that recruits Mec1 to sites of DNA damage. We isolated a ddc2 mutation that confers defects in DNA damage responses but does not impair Mec1 recruitment. We found that the catalytic activity of Mec1 increases in a Ddc2-dependent manner after DNA damage. Previous studies have demonstrated that Mec1 activation occurs through two independent pathways at G1 and G2/M: one pathway through Ddc1, a subunit of the checkpoint clamp and the second through Dpb11, the TopBP1 ortholog. We found that Mec1 activation occurs at least in part independently of Ddc1 and Dpb11. Our results suggest that Ddc2 stimulates Mec1 by a different mechanism than Ddc1 or Dpb11.
doi:10.1371/journal.pgen.1004136
PMCID: PMC3930518  PMID: 24586187
2.  Biochemical characterization of L1 repressor mutants with altered operator DNA binding activity 
Bacteriophage  2012;2(2):79-88.
A mycobacteriophage-specific repressor with the enhanced operator DNA binding activity at 32°C and no activity at 42°C has not been generated yet though it has potential in developing a temperature-controlled expression vector for mycobacterial system. To create such an invaluable repressor, here we have characterized four substitution mutants of mycobacteriophage L1 repressor by various probes. The W69C repressor mutant displayed no operator DNA binding activity, whereas, P131L repressor mutant exhibited very little DNA binding at 32°C. In contrast, both E36K and E39Q repressor mutants showed significantly higher DNA binding activity at 32°C, particularly, under in vivo conditions. Various mutations also had different effects on the structure, stability and the dimerization ability of L1 repressor. While the W69C mutant possessed a distorted tertiary structure, the P131L mutant dimerized poorly in solution at 32°C. Interestingly, both these mutants lost their two-domain structure and aggregated rapidly at 42°C. Of the native and mutant L1 repressor proteins, W69C and E36K mutants appeared to be the least stable at 32°C. Studies together suggest that the mutants, particularly P131L and E39Q mutants, could be used for creating a high affinity temperature-sensitive repressor in the future.
PMCID: PMC3442829  PMID: 23050218
mycobacteriophage L1; repressor; early promoter; operator DNA; mutant repressor and expression vector
3.  Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity 
Virology Journal  2007;4:64.
Background
Lysogenic mode of life cycle of a temperate bacteriophage is generally maintained by a protein called 'repressor'. Repressor proteins of temperate lambdoid phages bind to a few symmetric operator DNAs in order to regulate their gene expression. In contrast, repressor molecules of temperate mycobacteriophages and some other phages bind to multiple asymmetric operator DNAs. Very little is known at present about the structure-function relationship of any mycobacteriophage repressor.
Results
Using highly purified repressor (CI) of temperate mycobacteriophage L1, we have demonstrated here that L1 CI harbors an N-terminal domain (NTD) and a C-terminal domain (CTD) which are separated by a small hinge region. Interestingly, CTD is more compact than NTD at 25°C. Both CTD and CI contain significant amount of α-helix at 30°C but unfold partly at 42°C. At nearly 200 nM concentration, both proteins form appreciable amount of dimers in solution. Additional studies reveal that CI binds to O64 and OL types of asymmetric operators of L1 with variable affinity at 25°C. Interestingly, repressor – operator interaction is affected drastically at 42°C. The conformational change of CI is most possibly responsible for its reduced operator binding affinity at 42°C.
Conclusion
Repressors encoded by mycobacteriophages differ significantly from the repressor proteins of λ and related phages at functional level but at structural level they are nearly similar.
doi:10.1186/1743-422X-4-64
PMCID: PMC1934351  PMID: 17598887

Results 1-3 (3)