Bloom’s of BLM helicase may increase the risk of

Bloom’s syndrome is a
rare autosomal disorder that is caused due to mutations in BLM gene that is
located on chromosome 15 (1). The BLM gene encodes a 3′ to 5′ DNA helicase that
is important in the dissolution of Holliday Junction, G-quadruplexes and has
important roles in the 3 Rs (DNA Replication, Recombination and Repair) (2). The
absence of a functional BLM protein in patients with BLM syndrome compromise
the cell’s ability to maintain DNA integrity. This causes genome instability, elevated
levels of sister chromatid exchanges and homologous recombination and increased
predisposition to cancer (3).

Research on Bloom
syndrome has recently gained much momentum recently because several studies
indicate that BLM haploinsufficiency is associated with increased cancer risk. Study
by Goss et al (2002) established that
heterozygosity for a null mutation of BLM is associated with increased tumor
formation in mice (4). Similarly Gruber et
al (2002) showed that carriers of BLM mutations are linked to an increased
risk of developing colorectal cancer (5). Monoallelic BLM nonsense mutations
have also been shown to be associated with predisposition to breast cancer (6) (7).
Despite all these studies, it is not very clear whether normally occurring BLM
mutations that can cause minor defects in BLM helicase function might be a
cancer risk factor in normal healthy individuals.

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The study by Schmidt
and Mirzaei (2012) evaluates the
ability of SNPs in BLM gene to impair the functionality of the protein (8). A
follow up study showed that 3 SNPs, namely, R791C, P868L, G1120R showed some but not all phenotypes that is exhibited by
Bloom syndrome causing mutations. It was observed that these mutations are
associated with elevated levels of sister chromatid exchanges and improper DNA
damage response, but showed HU sensitivity and quadriradial chromosomes
comparable to wildtype. It is likely that the presence of these partial loss of
function alleles, that partially affect the function of BLM helicase may
increase the risk of cancer development (9).

The 1417 aminoacid long BLM protein contains 3 main domains: helicase
domain, RecQ- Ct domain and HRDC (Helicase, RNase D Conserved). All the three
domains are essential for function of the protein (10). The P868L mutation is
present in the C terminal lobe in ATPase domain and is present in a loop that
makes contact with double stranded DNA prior to unwinding. The G1120R is
present in a winged helix domain loop that mediates DNA binding. The R791C
mutation is present in N terminal lobe of ATPase domain and the phenotype
associated with this variant maybe due to reduced ATPase activity of the BLM
helicase (9).

Biochemical and functional analysis of all the above BLM variants will
be necessary to determine how exactly the mutation alters the function of the
helicase. The goal of my project was to express the above BLM variants in yeast
and to identify the clone that expresses high amounts of protein. This will be
followed up by a scale up of high protein expressing transformants,
purification of the protein using FPLC and biochemical assays like ATP
hydrolysis, helicase activity, double strand DNA binding to evaluate the
functionality of the protein.

 

 

 

METHODS

Plasmid Isolation

Transformation and
selection of E.Coli with plasmids
expressing the BLM variants of interest was performed by previous students in
the lab. The E.Coli strains
containing plasmids of interest was streaked out from glycerol stock to LB
ampicillin agar plates. Single colony of the strains were picked and inoculated
in 5ml LB media supplemented with 100mg/ml ampicillin. The bacterial cultures
were grown overnight at 37°C in a shaker. Plasmid isolation was performed using
GeneJETTM  Plasmid Miniprep
kit. Agarose gel electrophoresis was performed to validate plasmid isolation.

Yeast Transformation

For introduction of
plasmids into yeast, Lithium Acetate (LiAc) mediated transformation method was
used. Protease deficient yeast strain (4715) was used for this purpose. Yeast
competent cells were prepared in a solution containing LiAc, PEG and carrier
DNA and incubated at 30°C for 30 min. The cells were then heat shocked to
enable uptake of plasmid DNA and plated on synthetic dropout media lacking
uracil plates to select for transformants. At a cell density of 0.8, the
plasmid concentration was optimized to 2ng to obtain isolated single colonies.
Appropriate controls were used for transformation experiment

Selection of high
expressing clones

To select and screen
for yeast strains showing high protein expression, 7 single isolated colonies
were picked and inoculated in selection media. The transformants contain
plasmid with the BLM variant under galactose inducible promoter. When the cells
were in S-phase, protein expression was induced using 2% Galactose for 6h.  Protein isolation was performed using TCA
extraction protocol. Western Blotting was then carried out to identify which
clone expresses the highest amounts of BLM protein. 

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