36 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
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
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
Initiation of V(D)J recombination in a cell-free system
Cells performing V(D)J recombination make specific cuts in DNA at
recombination signal sequences. Here, we show that nuclear extracts of
pre-B cell lines carry out this specific cleavage. The products of
cleavage are the same as found previously in thymocytes: full-length,
blunt, 5'-phosphorylated signal ends, and covalently sealed (hairpin)
coding ends. A complete signal sequence is required. Recombinant RAG1
protein greatly increases activity and complements an inactive extract
from a RAG1 (-/-) pre-B cell line. When the extracts are fractionated,
cleavage activity correlates with the presence of RAG2 protein. These
results suggest that RAG1 and RAG2 are components of the V(D)J
recombinase
Homologous recombination deficiency testing for brca-like tumors: The road to clinical validation
Germline BRCA mutations result in homologous recombination deficiency (HRD) in hereditary breast and ovarian cancer, as well as several types of sporadic tumors. The HRD phenotype makes these tumors sensitive to DNA double strand break-inducing agents, including poly-(ADPribose)-polymerase (PARP) inhibitors. Interestingly, a subgroup of cancers without a BRCA mutation also shows an HRD phenotype. Various methods for selecting patients with HRD tumors beyond BRCA-mutations have been explored. These methods are mainly based on DNA sequencing or functional characteristics of the tumor. We here discuss the various tests and the status of their clinical validation
Role of the DNA damage response in prostate cancer formation, progression and treatment
Background: Clinical and preclinical studies have revealed that alterations in DNA damage response (DDR) pathways may play an important role in prostate cancer (PCa) etiology and progression. These alterations can influence PCa responses to radiotherapy and anti-androgen treatment. The identification of DNA repair gene aberrations in PCa has driven the interest for further evaluation whether these genetic changes may serve as biomarkers for patient stratification. Methods: In this review, we summarize the current knowledge on DDR alterations in PCa, their potential impact on clinical interventions and prospects for improved management of PCa. We particularly focus on the influence of DDR gene mutations on PCa initiation and progression and describe the underlying mechanisms. Results and Conclusions: A better understanding of these mechanisms, will contribute to better disease management as treatment strategies can be chosen based on the specific disease properties, since a growing number of treatments are targeting DDR pathway alterations (such as Poly(ADP-ribose) polymerase inhibitors). Furthermore, the recently discovered crosstalk between the DDR and androgen receptor signaling opens a new array of possible strategies to optimize treatment combinations. We discuss how these recent and ongoing studies will help to improve diagnostic, prognostic and therapeutic approaches for PCa management
Checkpoint kinase 2-mediated phosphorylation of BRCA1 regulates the fidelity of nonhomologous end-joining
The tumor suppressor gene BRCA1 maintains genomic integrity by protecting
cells from the deleterious effects of DNA double-strand breaks (DSBs).
Through its interactions with the checkpoint kinase 2 (Chk2) kinase and
Rad51, BRCA1 promotes homologous recombination, which is typically an
error-free repair process. In addition, accumulating evidence implicates
BRCA1 in the regulation of nonhomologous end-joining (NHEJ), which may
involve precise religation of the DSB ends if they are compatible (i.e.,
error-free repair) or sequence alteration upon rejoining (i.e.,
error-prone or mutagenic repair). However, the precise role of BRCA1 in
regulating these different subtypes of NHEJ is not clear. We provide here
the genetic and biochemical evidence to show that BRCA1 promotes
error-free rejoining of DSBs in human breast carcinoma cells while
suppressing microhomology-mediated error-prone end-joining and restricting
sequence deletion at the break junction during repair. The repair spectrum
in BRCA1-deficient cells was characterized by an increase in the formation
of >2 kb deletions and in the usage of long microhomologies distal to the
break site, compared with wild-type (WT) cells. This error-prone repair
phenotype could also be revealed by disruption of the Chk2 phosphorylation
site of BRCA1, or by expression of a dominant-negative kinase-dead Chk2
mutant in cells with WT BRCA1. We suggest that the differential control of
NHEJ subprocesses by BRCA1, in concert with Chk2, reduces the mutagenic
potential of NHEJ, thereby contributing to the prevention of familial
breast cancers