188 research outputs found
The RAG1 and RAG2 proteins establish the 12/23 rule in V(D)J recombination
V(D)J recombination requires a pair of signal sequences with spacer
lengths of 12 and 23 base pairs. Cleavage by the RAG1 AND RAG2 proteins
was previously shown to demand only a single signal sequence. Here, we
established conditions where 12- and 23-spacer signal sequences are both
necessary for cleavage. Coupled cutting at both sites require
Pathway choice in DNA double strand break repair:Observations of a balancing act
Proper repair of DNA double strand breaks (DSBs) is vital for the preservation of genomic integrity. There are two main pathways that repair DSBs, Homologous recombination (HR) and Non-homologous end-joining (NHEJ). HR is restricted to the S and G2 phases of the cell cycle due to the requirement for the sister chromatid as a template, while NHEJ is active throughout the cell cycle and does not rely on a template. The balance between both pathways is essential for genome stability and numerous assays have been developed to measure the efficiency of the two pathways. Several proteins are known to affect the balance between HR and NHEJ and the complexity of the break also plays a role. In this review we describe several repair assays to determine the efficiencies of both pathways. We discuss how disturbance of the balance between HR and NHEJ can lead to disease, but also how it can be exploited for cancer treatment.</p
Pathway choice in DNA double strand break repair:Observations of a balancing act
Proper repair of DNA double strand breaks (DSBs) is vital for the preservation of genomic integrity. There are two main pathways that repair DSBs, Homologous recombination (HR) and Non-homologous end-joining (NHEJ). HR is restricted to the S and G2 phases of the cell cycle due to the requirement for the sister chromatid as a template, while NHEJ is active throughout the cell cycle and does not rely on a template. The balance between both pathways is essential for genome stability and numerous assays have been developed to measure the efficiency of the two pathways. Several proteins are known to affect the balance between HR and NHEJ and the complexity of the break also plays a role. In this review we describe several repair assays to determine the efficiencies of both pathways. We discuss how disturbance of the balance between HR and NHEJ can lead to disease, but also how it can be exploited for cancer treatment.</p
Pathway choice in DNA double strand break repair: Observations of a balancing act
Proper repair of DNA double strand breaks (DSBs) is vital for the preservation of genomic integrity. There are two main pathways that repair DSBs, Homologous recombination (HR) and Non-homologous end-joining (NHEJ). HR is restricted to the S and G2 phases of the cell cycle due to the requirement for the sister chromatid as a template, while NHEJ is active throughout the cell cycle and does not rely on a template. The balance between both pathways is essential for genome stability and numerous assays have been developed to measure the efficiency of the two pathways. Several proteins are known to affect the balance between HR and NHEJ and the complexity of the break also plays a role. In this review we describe several repair assays to determine the efficiencies of both pathways. We discuss how disturbance of the balance between HR and NHEJ can lead to disease, but also how it can be exploited for cancer treatment
Specificity in V(D)J recombination: new lessons from biochemistry and genetics
Recent in vitro work on V(D)J recombination has helped to clarify its
mechanism. The first stage of the reaction, which can be reproduced with
the purified RAG1 and RAG2 proteins, is a site-specific cleavage that
generates the same broken DNA species found in vivo. The cleavage reaction
is closely related to known types of transpositional recombination, such
as that of HIV integrase. All the site specificity of V(D)J recombination,
including the 12/23 rule, is determined by the RAG proteins. The later
steps largely overlap with the repair of radiation-induced DNA
double-strand breaks, as indicated by the identity of several newly
characterized factors involved in repair. These developments open the way
for a thorough biochemical study of V(D)J recombination
Mutational analysis of the integrase protein of human immunodeficiency virus type 2
Purified integrase protein (IN) can nick linear viral DNA at a specific
site near the ends and integrate nicked viral DNA into target DNA. We have
made a series of 43 site-directed point mutants of human immunodeficiency
virus type 2 IN and assayed purified mutant proteins for the following
activities: site-specific cleavage of viral DNA (donor cut), integration
(strand transfer), and disintegration. In general, the different
activities were similarly affected by the mutations. We found three
mutations that (almost) totally abolished IN function: Asp-64-->Val,
Asp-116-->Ile, and Glu-152-->Leu, whereas 25 mutations did not affect IN
function. A few mutations affected the different activities
differentially. Near the amino terminus a zinc finger-like sequence motif
His-Xaa3-His-Xaa20-30-Cys-Xaa2-Cys is present in all retroviral IN
proteins. Two mutations in this region (His-12-->Leu and Cys-40-->Ser)
strongly inhibited donor cut but had less effect on strand transfer. The
central region of IN is most highly conserved between retroviral INs.
Three mutants in this region (Asn-117-->Ile, Asn-120-->Leu, and
Lys-159-->Val) were inhibited in strand transfer but were inhibited less
strongly in donor cut. Mutation of Asn-120 (to glycine, leucine, or
glutamate) resulted in changes in integration-site preference, suggesting
that Asn-120 is involved in interactions with target DNA. We did not find
a mutant in which one activity was lost and the others were unaffected,
supporting the notion that IN has only one active site for the catalysis
of donor cut and strand transfer
Identification of amino acids in HIV-2 integrase involved in site-specific hydrolysis and alcoholysis of viral DNA termini
The human immunodeficiency virus integrase (HIV IN) protein cleaves two
nucleotides off the 3' end of viral DNA and subsequently integrates the
viral DNA into target DNA. IN exposes a specific phosphodiester bond near
the viral DNA end to nucleophilic attack by water or other nucleophiles,
such as glycerol or the 3' hydroxyl group of the viral DNA molecule
itself. Wild-type IN has a preference for water as the nucleophile; we
here describe a class of IN mutants that preferentially use the 3'
hydroxyl group of viral DNA as nucleophile. The amino acids that are
altered in this class of mutants map near the putative active-site
residues Asp-116 and Glu-152. These results support a model in which
multiple amino acid side-chains are involved in presentation of the
(soluble) nucleophile. IN is probably active as an oligomeric complex, in
which the subunits have non-equivalent roles; we here report that
nucleophile selection is determined by the subunit that supplies the
active site
Identification of amino acids in HIV-2 integrase involved in site-specific hydrolysis and alcoholysis of viral DNA termini
The human immunodeficiency virus integrase (HIV IN) protein cleaves two nucleotides off the 3′ end of viral DNA and subsequently integrates the viral DNA into target DNA. IN exposes a specific phosphodiester bond near the viral DNA end to nucleophilic attack by water or other nucleophiles, such as glycerol or the 3′ hydroxyl group of the viral DNA molecule itself. Wild-type IN has a preference for water as the nucleophile; we here describe a class of IN mutants that preferentially use the 3′ hydroxyl group of viral DNA as nucleophile. The amino acids that are altered in this class of mutants map near the putative active-site residues Asp-116 and Glu-152. These results support a model in which multiple amino acid side-chains are involved in presentation of the (soluble) nucleophile. IN is probably active as an oligomeric complex, in which the subunits have non-equivalent roles; we here report that nucleophile selection is determined by the subunit that supplies the active site.</p
Identification of amino acids in HIV-2 integrase involved in site-specific hydrolysis and alcoholysis of viral DNA termini
The human immunodeficiency virus integrase (HIV IN) protein cleaves two nucleotides off the 3′ end of viral DNA and subsequently integrates the viral DNA into target DNA. IN exposes a specific phosphodiester bond near the viral DNA end to nucleophilic attack by water or other nucleophiles, such as glycerol or the 3′ hydroxyl group of the viral DNA molecule itself. Wild-type IN has a preference for water as the nucleophile; we here describe a class of IN mutants that preferentially use the 3′ hydroxyl group of viral DNA as nucleophile. The amino acids that are altered in this class of mutants map near the putative active-site residues Asp-116 and Glu-152. These results support a model in which multiple amino acid side-chains are involved in presentation of the (soluble) nucleophile. IN is probably active as an oligomeric complex, in which the subunits have non-equivalent roles; we here report that nucleophile selection is determined by the subunit that supplies the active site.</p
Exploiting DNA repair defects for novel cancer therapies
Most human tumors accumulate a multitude of genetic changes due to defects in the DNA damage response. Recently, small-molecule inhibitors have been developed that target cells with specific DNA repair defects, providing hope for precision treatment of such tumors. Here we discuss the rationale behind these therapies and how an important bottleneck-patient selection-can be approached
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