The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST is used to uncover several new and interesting members of the BRCT superfamily.

/pdf/gapped-blast-and-psi-blast-a-new-generation-of-protein-2ybl67x5yx.pdf

Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.

We report the identities of the members of a group of proteins that associate with BRCA1 to form a large complex that we have named BASC (BRCA1-associated genome surveillance complex). This complex includes tumor suppressors and DNA damage repair proteins MSH2, MSH6, MLH1, ATM, BLM, and the RAD50-MRE11-NBS1 protein complex. In addition, DNA replication factor C (RFC), a protein complex that facilitates the loading of PCNA onto DNA, is also part of BASC. We find that BRCA1, the BLM helicase, and the RAD50-MRE11-NBS1 complex colocalize to large nuclear foci that contain PCNA when cells are treated with agents that interfere with DNA synthesis. The association of BRCA1 with MSH2 and MSH6, which are required for transcription-coupled repair, provides a possible explanation for the role of BRCA1 in this pathway. Strikingly, all members of this complex have roles in recognition of abnormal DNA structures or damaged DNA, suggesting that BASC may serve as a sensor for DNA damage. Several of these proteins also have roles in DNA replication-associated repair. Collectively, these results suggest that BRCA1 may function as a coordinator of multiple activities required for maintenance of genomic integrity during the process of DNA replication and point to a central role for BRCA1 in DNA repair.

/pdf/basc-a-super-complex-of-brca1-associated-proteins-involved-36i2i4ir0k.pdf

BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures

The proteins encoded by the two major breast cancer susceptibility genes, BRCA1 and BRCA2, work in a common pathway of genome protection. However, the two proteins work at different stages in the DNA damage response (DDR) and in DNA repair. BRCA1 is a pleiotropic DDR protein that functions in both checkpoint activation and DNA repair, whereas BRCA2 is a mediator of the core mechanism of homologous recombination. The links between the two proteins are not well understood, but they must exist to explain the marked similarity of human cancer susceptibility that arises with germline mutations in these genes. As discussed here, the proteins work in concert to protect the genome from double-strand DNA damage during DNA replication.

BRCA1 and BRCA2: different roles in a common pathway of genome protection

The efficient repair of double-strand breaks in DNA is critical for the maintenance of genome stability and cell survival. Homologous recombination provides an efficient and faithful pathway of repair, especially in replicating cells, in which it plays a major role in tumour avoidance. Many of the enzymes that are involved in recombination have been isolated, and the details of this pathway are now being unravelled at the molecular level.

Molecular views of recombination proteins and their control

Dynamic Changes of BRCA1 Subnuclear Location and Phosphorylation State Are Initiated by DNA Damage

The hereditary breast and ovarian cancer gene, BRCA1, encodes a large polypeptide that contains the cysteine-rich RING motif, a zinc-binding domain found in a variety of regulatory proteins Here we describe a novel protein that interacts in vivo with the N-terminal region of BRCA1 This BRCA1-associated RING domain (BARD1) protein contains an N-terminal RING motif, three tandem ankyrin repeats, and a C-terminal sequence with significant homology to the phylogenetically conserved BRCT domains that lie near the C terminus of BRCA1 The BARD1/BRCA1 interaction is disrupted by BRCA1 missense mutations that segregate with breast cancer susceptibility, indicating that BARD1 may be involved in mediating tumour suppression by BRCA1

Identification of a RING protein that can interact in vivo with the BRCA1 gene product.

Germ-line mutations of the BRCA1 gene predispose women to early-onset breast and ovarian cancer by compromising the gene’s presumptive function as a tumor suppressor. Although the biochemical properties of BRCA1 polypeptides are not understood, their expression pattern and subcellular localization suggest a role in cell-cycle regulation. When resting cells are induced to proliferate, the steady-state levels of BRCA1 increase in late G1 and reach a maximum during S phase. Moreover, in S phase cells, BRCA1 polypeptides are hyperphosphorylated and accumulate into discrete subnuclear foci termed “BRCA1 nuclear dots.” BRCA1 associates in vivo with a structurally related protein termed BARD1. Here we show that the steady-state levels of BARD1, unlike those of BRCA1, remain relatively constant during cell cycle progression. However, immunostaining revealed that BARD1 resides within BRCA1 nuclear dots during S phase of the cell cycle, but not during the G1 phase. Nevertheless, BARD1 polypeptides are found exclusively in the nuclear fractions of both G1- and S-phase cells. Therefore, progression to S phase is accompanied by the aggregation of nuclear BARD1 polypeptides into BRCA1 nuclear dots. This cell cycle-dependent colocalization of BARD1 and BRCA1 indicates a role for BARD1 in BRCA1-mediated tumor suppression.

https://www.pnas.org/content/pnas/94/22/12075.full.pdf

Cell cycle-dependent colocalization of BARD1 and BRCA1 proteins in discrete nuclear domains

Metabolic engineering for Escherichia coli BW25113 for the production of 5-Aminolevulinic Acid based on CRISPR/Cas9 mediated gene knockout and metabolic pathway modi cation

Meng-Chun W. Yang

Papers

Identification of a RING protein that can interact in vivo with the BRCA1 gene product.

Cell cycle-dependent colocalization of BARD1 and BRCA1 proteins in discrete nuclear domains

Metabolic engineering for Escherichia coli BW25113 for the production of 5-Aminolevulinic Acid based on CRISPR/Cas9 mediated gene knockout and metabolic pathway modi cation