Jamie Knox
11/8/07
Honors Genetics
The Genetic Basis of Bloom’s Syndrome
Bloom’s Syndrome (BS) is an autosomal recessive condition presenting with a
variety symptoms, the most prevalent being growth defects, fertility problems,
immunodeficiency, and cancer predisposition (Guo et al, 2007). In the cell, BS results in
chromosomal instability and an abnormally high rate of chromosomal missegregation and
dysfunction (Chan et al, 2007). This disorder can be recognized by a sister chromatid
exchange rate equivalent to ten times the normal rate in the cell. A sister chromatid
exchange involves the trade of homologous DNA sequences between sister chromatids
which occurs normally during mitosis but at an elevated rate following DNA damage.
Sister chromatid exchanges are thought to be a part of the cellular repair response to
problems such as the disruption of DNA replication forks (Chan et al, 2007).
The BS phenotype can be attributed to mutations in BLM, a gene coding for a
DNA helicase enzyme from the RecQ family (Otsuki et al, 2007). As a helicase, the
BLM protein functions to separate DNA strands during DNA replication and related
processes (Guo et al, 2007). When complexed with topoisomerase III alpha, BLM
protein also serves to resolve an intermediate step in the process of homologous
recombination known as double Holliday junctions (Otsuki et al, 2007, Plank et al,
2006). Besides its role in the maintenance of genetic diversity, homologous
recombination also plays a part in DNA repair and the regeneration of DNA replication
forks, two processes with which BLM is associated (Plank et al, 2006). While BLM-
related data has been building up in recent years, relatively little was known about the
protein’s molecular function. Recently, several studies have been performed which
examine BLM protein’s role in mitosis, position in recombination and damage response
pathways, and the functional result of particular mutations within the BLM DNA
sequence.
Chan et al. (2007) published a study concerning the separation of chromosomes
during anaphase and the role of BLM protein in that process. Researchers used two cell
lines for the study; PSNF5 cells, which express the BLM protein, and PSNG13 cells,
which do not. Examination of a 600-cell sample which included both of these cell lines
in anaphase found a significantly larger frequency of chromosome segregation problems
in PSNG13 cells as compared to PSNF5 cells. These problems included both anaphase
bridges and lagging chromatin (see picture below), implying that BLM function is
necessary for proper chromosome segregation.
Graph (A) represents results from study of chromosome segregation problems. Pictures (B) represent
examples of anaphase bridges (first 3 pictures) and lagging chromatin (last picture). (Chan et al 2007)
The second part of the study performed by Chan et al. (2007) examined the
presence of BLM protein at anaphase bridges in cells from a functionally normal human
fibroblast line (GM00637 cells). Cells were stained using anti-BLM antibodies and
immunofluorescence images were obtained. It was found that over 95% of cells
containing an anaphase bridge had BLM protein associated with that same area. Besides
supporting the hypothesis that completion of chromosome segregation is dependent upon
BLM, this study revealed the presence of “ultrafine bridge-like structures” between the
daughter nuclei of a number of cells. These structures could also be found between
lagging chromatin and the cluster of DNA from which the chromatin was separated.
They were termed BLM-DNA bridges since they stained for BLM and were confirmed to
be comprised of DNA. Interestingly, the researchers also noticed that BLM-DNA
bridges existed in anaphase cells which appeared normal and lacked anaphase bridges.
The final section of the study performed by Chan et al. (2007) focused on the
incidence of both anaphase bridges and BLM-DNA bridges during the course of
anaphase using the normal human fibroblast cell line. In this study, 10% of the total cells
were in early anaphase, meaning that the two clusters of chromatin were close together,
while 90% of the total cells were in late anaphase, meaning the chromatin clusters were
near the opposite poles of the cell. It was found that 89% of the early anaphase cells
contained one of the two bridge structures, a surprising statistic since these cells were
functionally normal. In late anaphase, 42% of the cells still had one of the bridge
structures. It was also determined that the frequency of BLM-DNA bridges could be
altered by inhibiting a part of the BLM protein’s pathway. Taken together, these data
show that cells which fail to express BLM, display a greater frequency of bridge
structures, yet bridge structures are more common in normal cells than researchers had
previously believed. As normal cells progress through anaphase, bridge structures tend to
disappear, indicating that BLM is responsible for correcting the anaphase and BLM-DNA
bridges. Dysfunctional BLM results in improper chromosome segregation and
chromosomal defects, as displayed by Bloom’s Syndrome.
Otsuki et al (2007) investigated BLM function in relation to other cellular
response proteins in an effort to better understand some of the processes completed by
BLM in the cell. Using DT40 cells from a chicken, researchers created and studied
mutant cells having two and three mutations including mutation of BLM and related
repair-pathway proteins. The protein named XRCC3, which plays a part in homologous
recombination, was found to work closely with BLM. Looking at various combinations
of mutant cells, Otsuki et al (2007) found that increased levels of sister chromatid
exchanges in BLM mutant cells could be attributed to XRCC3 function. Through
inhibition of topoisomerase III alpha, the other member of the BLM complex which
resolves double Holliday junctions, cells were found to have an increased rate of sister
chromatid exchanges. Inhibition of topoisomerase III alpha in BLM mutant cells resulted
in a sister chromatid exchange rate nearly identical to that of regular BLM mutant cells,
indicating that BLM and topoisomerase III alpha function together in the prevention of
sister chromatid exchange formation.
Otsuki et al (2007) also considered various chromosomal problems in different
combinations of BLM and XRCC3 mutants resulting from exposure to methyl
methanesulfonate and ultraviolet light, two DNA damaging agents. They found that
XRCC3 expression in BLM mutant cells results in a decreased response to methyl
methanesulfonate and ultraviolet light, indicating that functional XRCC3 is responsible
for the sensitivity of BS cells to these two damaging agents. Based on their results,
Otsuki et al (2007) concluded that XRCC3 and BLM exist in the same DNA damage
response pathway in the cell and that XRCC3 precedes BLM in that pathway.
Guo et al (2007) investigated five mutations in the core of the BLM protein.
They decided to use the protein fragment BLM642-1290 of the BLM protein in their studies
because its smaller size makes it easier to analyze and it functions like the complete BLM
protein in that it resolves double Holliday junctions and prevents damage-response
recombination in cells exposed to ultraviolet light. Using this protein fragment,
researchers tested the ability of the mutations to affect protein functions such as helicase,
ATPase, DNA binding ability, and the protein’s strand annealing ability (ability to repair
double strand breaks occurring between two identical sequences). The following table
summarizes their findings, with the BLM row being activity of the wild type protein and
the other five rows being the enzymatic activity in relation to that of the wild type
protein:
The enzymatic activity of BLM mutants. (Guo et al 2007)
Results show that helicase and ATPase function is nearly nonexistent in all five mutant
types. Mutants Q672R and I841T display a nearly normal amount of enzymatic activity
in the area of DNA binding, indicating that they do not play a significant part in the
process. Mutant Q672R is the only mutation which does not show nearly normal
enzymatic function in regard to ATP binding, indicating that it is involved in the binding
of ATP.
These studies have better characterized the niche of BLM in the cell and the
underlying causes of Bloom’s Syndrome. Chan et al. (2007) found that BLM-deficient
cells fail to properly complete anaphase due to unresolved anaphase bridges and BLMDNA bridges, resulting in chromosome segregation problems. Otsuki et al (2007)
studied the function of BLM in homologous recombination and DNA damage response
pathways in the cell, finding that the BLM / topoisomerase III alpha complex is directly
responsible for the suppression of sister chromatid formation. Guo et al (2007) worked
with specific mutations in five different areas of the BLM DNA sequence and
characterized the effect of each on normal BLM function. Further studies will improve
our understanding of the cellular function of BLM.
Works Cited
Chan, Kok-Lung, Phillip S. North, Ian D. Hickinson (2007).BLM is required for faithful
chromosome segregation and its localization defines a class of ultraphine
anaphase bridges. The EMBO Journal. 26, 3397-3409.
Guo, Rong-Bing, Pascal Rigolet, Hua Ren, Bo Zhang, Xing-Dong Zhang, Shuo-Xing
Dou, Peng-Ye Wang, Mounira Amor-Gueret and Xu Guang Xi (2007). Structural
and functional analyses of disease-causing missense mutations in Bloom
Syndrome Protein Nucleic Acids Research. 35, 6297-6310.
Otsuki, Makoto, Masayuki Seki, Eri Inoue, Akari Yoshimura, Genta Kato, Saki
Yamanouchi, Yoh-ichi Kawabe, Shusuke Tada, Akira Shinohara, Jun-ichiro
Komura, Tetsuya Ono, Shunichi Takeda, Yutaka Ishii, and Takemi Enomoto
(2007). Functional interactions between BLM and XRCC3 in the cell. Journal of
Cell Biology. 179, 53-63.
Plank, Jody L., Tao-shih Hsieh (2006). A novel, topologically constrained DNA
molecule containing a double Holliday junction: design, synthesis, and initial
biochemical characterization. Journal of Biological Chemistry. 281, 1751017516.