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Search results for: Uracil DNA Glycosilase

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#25483076   // To Up

Checkpoint kinase 2 is required for efficient immunoglobulin diversification.

Maintenance of genome integrity relies on multiple DNA repair pathways as well as on checkpoint regulation. Activation of the checkpoint kinases Chk1 and Chk2 by DNA damage triggers cell cycle arrest and improved DNA repair, or apoptosis in case of excessive damage. Chk1 and Chk2 have been reported to act in a complementary or redundant fashion, depending on the physiological context. During secondary immunoglobulin (Ig) diversification in B lymphocytes, DNA damage is abundantly introduced by activation-induced cytidine deaminase (AID) and processed to mutations in a locus-specific manner by several error-prone DNA repair pathways. We have previously shown that Chk1 negatively regulates Ig somatic hypermutation by promoting error-free homologous recombination and Ig gene conversion. We now report that Chk2 shows opposite effects to Chk1 in the regulation of these processes. Chk2 inactivation in B cells leads to decreased Ig hypermutation and Ig class switching, and increased Ig gene conversion activity. This is linked to defects in non-homologous end joining and increased Chk1 activation upon interference with Chk2 function. Intriguingly, in the context of physiological introduction of substantial DNA damage into the genome during Ig diversification, the 2 checkpoint kinases thus function in an opposing manner, rather than redundantly or cooperatively.
Kathrin Davari, Samantha Frankenberger, Angelika Schmidt, Nils-Sebastian Tomi, Berit Jungnickel

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#10342105   // To Up

Simplified PCR set-up using a frozen preformulated mix for the detection of cytomegalovirus.

A simple PCR set-up for the detection of cytomegalovirus in clinical specimen was developed. All components of the PCR master mix including Taq DNA polymerase, uracil N-glycosilase, and primers were preformulated and stored frozen in aliquots. After thawing the master mix aliquots, the PCR was immediately started after the addition of sample DNA. This method gave excellent reproducible PCR-results without loss of enzyme activities following storage at -20 degrees C for at least 4 months.
B Kofler, A Klausegger

2231 related Products with: Simplified PCR set-up using a frozen preformulated mix for the detection of cytomegalovirus.



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#9208173   // To Up

New strategy for the construction of single-stranded plasmids with single mutagenic lesions.

Single-stranded DNA vectors containing single adducts offer a unique opportunity to study the biochemistry and genetics of trans lesion synthesis, a process during which a DNA polymerase synthesizes across a lesion. We describe a new and general strategy to produce high-quality single-stranded plasmids containing a single adduct within a predetermined sequence context starting with a short oligonucleotide containing the lesion of interest. These vectors are isolated from the corresponding double-stranded constructs by selective enzymatic degradation in vitro of the nonadducted uracil-containing strand. Efficient and complete removal of this strand was achieved using uracil DNA glycosilase to generate AP sites followed by the action of the AP endonuclease associated with exonuclease III and the robust 3'-->5' exonuclease activity associated with T7 DNA polymerase. We show the utility of these constructs for the study of trans lesion synthesis in vitro and in vivo in the case of the highly carcinogenic N-2-acetylaminofluorene adducts located within frameshift mutation hot spots. The possibility to construct both single-stranded and double-stranded plasmids, with the same origin of replication (i.e., ColE1), will allow a direct comparison between single-stranded and double-stranded DNA replication in site-specific mutagenesis studies.
R L Napolitano, R P Fuchs

1581 related Products with: New strategy for the construction of single-stranded plasmids with single mutagenic lesions.

200 ug100 ug100 ug5 x 200 ug200 units5 x 200 ug200 units200 ug5 mg1ml5 g

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