Environmental Factor, May 2010, National Institute of Environmental Health Sciences
DNA Strand Break Repair and Human Disease
By Omari J. Bandele
On April 13, Steve West, Ph.D., of the London Research Institute in the United Kingdom, presented "Defects in DNA Strand Break Repair and Links to Human Disease," a talk hosted by NIEHS Staff Scientist Dmitry Gordenin, Ph.D.
The seminar was the latest in the NIEHS Distinguished Lecture Series. West's research involves understanding how flaws in basic DNA repair processes - such as homologous recombination - contribute to tumorigenesis.
Early in his presentation, West explained that cells are constantly bombarded with a wide variety of DNA damage, including DNA strand breaks and cross-links, alkylation lesions, and base loss. "The maintenance of genome integrity is essential to all organisms," West emphasized. "Cells have elegant mechanisms that can specifically recognize and repair different types of DNA damage."
West underscored the vital role of DNA repair by observing that defects in repair processes are associated with many human cancers, such as xeroderma pigmentosum, hereditary non-polyposis colorectal cancer (HNPCC), Fanconi anemia, and breast cancer.
BRCA2 in the repair of double-strand DNA breaks
In his talk, West illustrated the contribution of the BRCA2 tumor suppressor and the Rad51 recombinase to the repair of double-strand DNA breaks via homologous recombination. He stated that approximately 10 percent of genetically linked breast cancers - the second leading cause of cancer in women - involve a mutated version of the BRCA2 gene. Defects in BRCA2 are associated with decreased DNA repair and elevated levels of chromosome instability, which can led to tumorigenesis.
To further investigate the role of BRCA2 in repairing double-strand DNA breaks, West and colleagues successfully purified the full-length protein - a feat that had not previously been accomplished. The researchers observed extensive Rad51 binding to the intact protein. They also revealed that BRCA2 bound only single-stranded DNA.
Identification of eukaryotic nucleases that resolve Holliday junctions
The removal of Holliday junctions (HJ) is paramount for proper chromosome segregation during homologous recombination-dependent DNA repair. After searching nearly 20 years, West and colleagues identified two eukaryotic HJ resolvases - Yen1 in yeast and GEN1 in humans. These proteins represent a new subclass of the Rad2/XPG nuclease family, which resolves HJs in a manner analogous to the bacterialresolvase, RuvC.
West explained that yeasts utilize Yen1 and the Mus81-EME1 nuclease complex to remove HJ that form during meiosis or following treatments that cause replication blockage. Cells defective in both Mus81 and Yen showed a severe phenotype indicating that toxic recombination intermediates accumulate in the absence of Yen1 and Mus81-EME1. He also proposed that Yen1 acts to remove intermediates that escape detection by the Mus81-EME1 complex.
GEN1, Mus81, and BLM proteins play distinct roles in homologous recombination
West believes junction dissolution - unlike junction resolution - is a safer way to remove HJ in human mitotic cells. Junction dissolution is mediated by the BLM protein complex and involves DNA decatentation reactions. "This may be the primary mechanism to avoid potentially cytotoxic sister chromatid exchanges (SCE) in mitotic cells," West noted.
Cells from patients with Bloom syndrome - characterized by the lack of BLM helicase activity - have high levels of SCE and genome instability due to the absence of the junction dissolution pathway. As a result, the GEN1 and Mus81-EME1 resolvase-mediated junction-resolution pathways predominate.
To determine the role of GEN1 and the Mus81-EME1 complex in SCE during DNA repair, West and colleagues examined Holliday junction resolution in BLM-deficient cells in the absence of GEN1, Mus81, or both proteins. They observed reduced SCE in cells that lacked Mus81, whereas no obvious changes were seen in the absence of GEN1. West suggests that these results indicate that the Mus81-EME1 complex is primarily responsible for SCE formation.
West concluded his presentation by illustrating that cells which lack all three pathways - BLM, GEN1, and Mus81-EME1 - display "chromosome catastrophe" characterized by extensive chromosome fragmentation. West's work provides evidence that the removal of HJ is critical for the maintenance of genomic stability and, ultimately, protection against tumorigenesis.
(Omari J. Bandele, Ph.D., is a postdoctoral fellow in the NIEHS Laboratory of Molecular Genetics Environmental Genomics Group.)