DNA Repair-HR

Homologous Recombination is a genetic recombination in which two similar or identical double-stranded

or single-stranded nucleic acids exchange their nucleotide sequence. It is mostly used to repair breaks
known as double-strand breaks (DSB) in DNA.

Double strand repair in DNA occur through the following steps:

i. double-strand break occurs and then sections of DNA around the 5' ends of the break are cut
away in a process called resection.
ii. then strand invasion step follows, an overhanging 3' end of the broken DNA molecule then
invade a similar or identical DNA molecule that is not broken.
iii. Then further sequence follows either of the known pathways:

(a)dHJ or DSBR Pathway (b) SDSA Pathway (c)SSA Pathway (d)BIR pathway

Of them DSBR and SDSA are the main-

First processes are same for both:

MRX complex (Mre11-Rad50-Xrs2) protein binds to DNA on either side of the break.
Next step involves resection, which is done in two steps. In the first step of resection, the
MRX complex recruits the Sae2 protein. The two proteins then trim back the 5' ends on
either side of the break to create short 3' overhangs of single-strand DNA. And in the
second step, 5'→3' resection is continued by the Sgs1 helicase and
the Exo1 and Dna2 nucleases. As a helicase, Sgs1 "unzips" the double-strand DNA,
while Exo1 and Dna2's nuclease activity allows them to cut the single-stranded DNA
produced by Sgs1. RPA protein then binds to the 3’ overhangs due to its high affinity to
single-stranded DNA. With the help of several other proteins that mediate the process,
the Rad51 protein then forms a filament of nucleic acid and protein on the single strand of
DNA coated with RPA. This nucleoprotein filament then begins searches for DNA
sequences similar to that of the 3' overhang. After finding such a sequence, the single-
stranded nucleoprotein filament invades the similar or identical recipient DNA duplex. A
displacement loop (D-loop) is formed during strand invasion between the invading 3'
overhang strand and the homologous chromosome. Then, a DNA polymerase extends
the end of the invading 3' strand by synthesizing new DNA. This changes the D-loop to a
cross-shaped structure known as a Holliday junction. DNA synthesis occurs on the
invading strand, also restoring the strand on the homologous chromosome that was
displaced during strand invasion.

Difference:

(a) Double Holliday Junction(dHJ) pathway or Double strand bond repair (DSBR)
pathway:
In DSBR pathway, the 3’ overhang which was not involved in the strand invasion also
forms a Holliday junction with the homologous chromosome. Both of the Holliday
junctions are then converted into recombination products by nicking endonucleases.
The DSBR pathway mostly results in crossover products but also gives non-
crossover outcomes sometimes.

(b) Synthesis-dependent strand annealing (SDSA) pathway:

 In SDSA pathway, the 3’ invading strand is extended along the recipient DNA duplex
by a DNA polymerase, and is then released as the Holliday junction between the
donor and recipient DNA molecules slides in a process called branch migration. The
newly synthesized 3' end of the invading strand is then to anneals to the other 3'
overhang in the damaged chromosome through complementary base pairing. After
the strands anneal, a small section of DNA may remain. These remains are removed,
and the SDSA pathway ligation of any remaining single-stranded gaps. The SDSA
pathway results non-crossover outcome with no change in template DNA.
 SSA Pathway
This pathway takes place when resection reveals flanking homologous repeats that can anneal,
leading to deletion of the intervening sequences. the SSA pathway only requires a single DNA
duplex, and uses the repeat sequences as the identical sequences that homologous
recombination needs for repair. This pathway follows a simple idea: two strands of the same DNA
duplex are cut back around the site of the double-strand break, the two resulting 3' overhangs
then align and anneal to each other, restoring the DNA as a continuous duplex.

 BIR Pathway
Double-strand breaks sometimes are seen at replication forks as DNA helicase unzips the
template strand. These defects are repaired in the break-induced replication (BIR) pathway.
BIR involves both leading and lagging strand synthesis and hence results in the loss of
heterozygosity or, if the template is located ectopically, a nonreciprocal translocation.

 HR maintains genomic stability in mammalian mitotic cells through precise templated repair of
DNA double strand breaks and other lesions that are encountered during normal cellular
metabolism and from exogenous insults. Hence homologous combination repair is essential
during proliferative stages in development and during somatic cell renewal in adults to protect
against cell death and mutagenic outcomes from DNA damage. Mutations in mammalian gene
encoding homologous recombination proteins are associated with developmental abnormalities
and tumorigenesis.
 Integration of a DNA fragment in a host genome requires the action of a double‐strand break
repair mechanism. HR is initiated by binding of Rad52p to DNA ends and results in targeted
integration. Binding of the Ku heterodimer (Ku70p/Ku80p) results in random integration via non‐
homologous end joining (NHEJ).  The model fungus Saccharomyces cerevisiae  has an extremely
efficient homologous recombination (HR) mechanism which results in a very high targeted gene
deletion frequency. In contrast to Saccharomyces cerevisiae, the budding yeast Kluyveromyces
lactis  shows variable, but in general low, gene targeting efficiency. To study and to improve gene
targeting efficiency, K. lactis has been used as a model. The KlRAD51,
KlRAD52 and KlKU80  genes have been isolated and deletion mutants for these genes have
been constructed.  In wild‐type K. lactis, it was seen that the gene targeting efficiency ranged
from 0% with 50 to 88% with 600 bp flanks. While the Klku80 mutant showed >97% gene
targeting efficiency independently of the size of the homologous flanks.  Efficiency of gene
targeting was determined at the KlADE2 locus using targeting constructs with different lengths of
homologous flanking sequences. The results demonstrated that deletion of the NHEJ mechanism
results in a higher gene targeting efficiency. Furthermore, increased gene targeting efficiency was
achieved by the transformation of wild‐type K. lactis  with the KlADE2  deletion construct in the
presence of excess small DNA fragments. Using this method, PCR‐generated deletion constructs
containing only 50 bp of homologous flanking sequences resulted in efficient targeted gene
replacement. 
RESTRICTION AND LIGATION-BASED CLONING

Restriction and ligation-based cloning is one of the simplest methods of cloning. It begins with preparing a vector to
receive an insert DNA by digesting each with restriction enzymes. And then the digested fragments are spliced
together by ligases.

Process:

Vectors used here are plasmids, which are double stranded circular DNAs that replicate inside bacteria independently
of the genomic DNA. All cloning vectors based on plasmids contain a number of elements, including a bacterial origin
of replication to efficiently propagate within the bacterial host cell; a multiple cloning site (MCS) that contains a
number of restriction enzyme sites to allow ready addition of an DNA insert of interest; and a marker to select for
bacteria after successful uptake of the vector.

Restriction enzymes (restriction endonucleases) cut DNA at (or close to) specific recognition sites and there are two
types:

i. Blunt end cutters. These enzymes cut both strand of the target DNA at the same spot creating blunt
ends.
ii. Sticky end cutters. These enzymes cut both strand of the target DNA at different spots creating 3'- or
5'-overhangs of 1 to 4 nucleotides (so-called sticky ends).

To be able to clone a DNA insert into a cloning or expression vector, both have to be treated with two restriction

enzymes that create compatible ends. At least one of the enzymes used should be a sticky end cutter to ensure that

the insert is incorporated in the right orientation.

The next step is the ligation of the insert into the linearized vector. This involves the formation of phosphodiester

bonds between adjacent 5'-phosphate and 3'-hydroxyl residues, which can be catalyzed by two different ligases: E.

coli DNA ligase and bacteriophage T4 DNA ligase.

Experiment:

HLA class I alleles are recognized for their protective effect on HIV-1 pathogenesis and
disease progression. It is due to their ability to target conserved portions of the HIV-1
genome that escape with difficulty. Sequence changes attributed to cellular immune
pressure arise across the genome during infection, and if found within conserved
regions of the genome such as Gag, can affect the ability of the virus to replicate in
vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients
has been associated with reducing viral loads. This may be due to a reduced replication
capacity of the virus. Hence to study this, in vitro replication of HIV-1 as influenced by
the gag gene was observed. The gag gene was isolated from acute time points from
subtype C infected Zambians and were inserted to common subtype C HIV-1 proviral
backbone (MJ4) using restriction enzyme-based cloning. This makes it more appropriate
to the study of subtype C sequences than previous recombination-based methods that
have assessed the in vitro replication of chronically derived gag-pro sequences.
Nevertheless, the protocol could be voluntarily modified for studies of viruses from
other subtypes. Furthermore, this protocol details a robust and reproducible method for
assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T
cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived
from 149 subtype C acutely infected Zambians, and has allowed for the identification of
residues in Gag that affect replication. More importantly, the implementation of this
technique has facilitated a deeper understanding of how viral replication defines
parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal
CD4+ T cell decline.

appropriate