RECAP: Questions based on previous lectures

Explain the following:

1.Failure to repair DNA damage may lead to either mutation
or cell death
2.Single stranded DNA damage is easy to repair
3.The mismatch repair system involves multiple enzymes
4.Repair systems of double stranded DNA breaks are more
error-prone
5.Recombination may lead to major changes in the genome
of a cell
6. Mutational inactivation of genes encoding DNA repair
systems should cause major disadvantage for the
survival
of a cell
RECAP: Questions based on previous lecture
State True or False:

1.A point mutation refers to insertion of a stretch of nucleotides

at a particular point in the DNA
2.An open reading frame refers to the nucleotide sequence
bound by a start codon and an stop codon
3.Homologous recombination refers to recombination between
two DNA fragments of nearly identical sequence
4. Recombination can not restore the wild type sequence of a
mutated gene
5.An excision repair involves both exonuclease and polymerase
activities of repair enzymes
 Genetic Recombination

Lecture 11: Expected Learning Outcome (ELO)

1. Definition of genetic recombination
2. Type of genetic recombination
3. Consequence of recombination
 Genetic Recombination as a
means of genetic variation
Breaking and rejoining of two parental
DNA molecules to produce new DNA
molecules
Recombination occurs when two homologous
chromosomes are together
• Homologous or general recombination:
– Bacterium with two viruses
– Bacterium after conjugal transfer of part of a
chromosome
– During meiosis of eukaryotic cells
– Post-replication repair via retrieval system
• Other types of recombination
– Site specific : Integration of bacterial, viral or
plasmid DNA into cellular chromosome
– Replicative : Transposition
 Recombination
• Breaking and rejoining of two parental DNA
molecules to produce new DNA molecules
• Reciprocal recombination: new DNA
molecules carry genetic information from
both parental molecules.
• Gene conversion: one way transfer of
information, resulting in an allele on one
parental chromosome being changed to the
allele from the other homologous
chromosome
 Different types of naturally occurring recombination have
been identified in living organisms.
(a) Homologous (b) Non-homologous
(c ) Site-directed and (d) Replicative

General or homologous recombination occurs between

DNA molecules of very similar sequence, such as
homologous chromosomes in diploid organisms. General
recombination can occur using one or a small number of
common enzymatic pathways.
A+ B+ C+ A+ B- C-
Homologous
or general A- B- C- A- B+ C+

Each line represents a chromosome or segment of a chromosome; thus a single line

represents both strands of duplex DNA. Recombination between genes A and B leads
to a reciprocal exchange of genetic information, changing the arrangement of alleles
on the chromosomes.
Recombination is central to many biological processes
such as the generation of genetic variation (and therefore
evolution) and the incorporation of viral DNA into host
DNA, resulting in successful infection.

Homologous recombination is a type of recombination in

which nucleotide sequences are exchanged between two
similar or identical molecules of DNA.

The process of DNA recombination occurs in distinct

stages with the formation of a four-way Holliday junction
being a central step.
For the reaction to proceed the Holliday
junction must be cleaved to yield 2 linear
duplexes. This process is regulated by a
junction-resolving enzyme that cleaves the
Holliday junction resulting in rearranged DNA
strands.

For example, bacteriophage T7 encodes a

protein, endonuclease I, which acts as a
Holliday junction resolving enzyme during
integration into the bacterial chromosome.
 Holliday Junction
A Holliday junction is a
branched nucleic acid structure
that contains four double-
stranded arms joined together.
The structure is named after the
molecular biologist Robin
Holliday, who proposed its
existence in 1964.
For recombination to proceed
the 4 stranded Holliday
junction is cleaved resulting in
two rearranged DNA duplexes.
Holliday junctions are a key intermediate in many types of genetic recombination,
as well as in double-strand break repair. These junctions usually have a
symmetrical sequence and are thus mobile, meaning that the four individual arms
may slide through the junction in a specific pattern that largely preserves base
pairing.
 Holliday Junction

• The Holliday junction is the area of cross-over

that occurs during recombination
Genetic Recombination
 The recA Enzyme
The RecA protein is a central component in the processes
of homologous genetic recombination in Escherichia coli.
Functional homologs of RecA have been identified in
every organism examined.
RecA is a DNA-dependent ATPase that catalyzes a strand
exchange reaction between homologous DNA molecules.
The active form of the protein is a nucleoprotein filament
formed when RecA monomers polymerize onto single-
stranded DNA.
In addition to its direct role in recombinational processes,
the RecA protein regulates other repair pathways by
mediating the induction of the E. coli SOS response to
excessive DNA damage.
 RecBCD Enzyme Complex
RecBCD, an enzyme of E. coli initiates recombinational
repair from potentially lethal double strand DNA breaks.
The RecBCD enzyme is both a helicase that unwinds, or
separates the strands of DNA, and a nuclease that makes
single-stranded nicks in DNA. The enzyme complex is
composed of three different subunits called RecB, RecC,
and RecD and hence the complex is named RecBCD

gene chain protein function

RecB beta P08394 3'-5' helicase, nuclease
Likely recognizes Chi (crossover
RecC gamma P07648
hotspot instigator)
RecD alpha P04993 5'-3' helicase
Illegitimate or nonhomologous recombination occurs in
regions where no large-scale sequence similarity is apparent,
e.g. translocations between different chromosomes or
deletions that remove several genes along a chromosome.
However, when the DNA sequence at the breakpoints for
these events is analyzed, short regions of sequence similarity
are found in some cases. For instance, recombination
between two similar genes that are several million bp apart
can lead to deletion of the intervening genes in somatic cells.
A B C A B F
Nonhomologous
or illigitimate C
D E F D E

Two different DNAs recombine, moving, e.g. gene C so that it is now

on the same chromosome as genes D and E. Although the sequences
of the two chromosomes differ for most of their lengths, the
segments at the sites of recombination may be related, denoted by
the yellow and orange rectangles.
Site-specific recombination occurs between particular short
sequences (about 12 to 24 bp) present on otherwise
dissimilar parental molecules. Site-specific recombination
requires a special enzymatic machinery. For example the
integration of some phage genomes in bacterial chromosome

 att  integrase

Site-specific
att att
att E. coli

Site-specific recombination leads to the combination of two

different DNA molecules, illustrated here for a bacteriophage
integrating into the E. coli chromosome. This reaction is
catalyzed by a specific enzyme that recognizes a short sequence
present in both the phage DNA and the target site in the
bacterial chromosome, called att.
The third type is replicative recombination, which
generates a new copy of a segment of DNA. Many
transposable elements use a process of replicative
recombination to generate a new copy of the
transposable element at a new location.

transposase
A B C A B C

Transposable element

Replicative recombination is seen for some transposable

elements, shown as red rectangles, again using a specific
enzyme, in this case encoded by the transposable element.
 Types of recombination
A+ B+ C+ A+ B- C-
Homologous
or general A- B- C- A- B+ C+

A B C A B F
Nonhomologous
or illigitimate C
D E F D E

 att  integrase

Site-specific
att att
att E. coli

Replicative transposase
A B C A B C
recombination,
transposition
Transposable element
 Gene Conversion

A+ B+ C+ A+ B+ C+

A- B- C- A- B+ C-
Mechanisms of Homologous Recombination
During unwinding of DNA, the nuclease in RecB can
act in different ways depending on the reaction
conditions, notably the ratio of the concentrations of
Mg2+ ions and ATP. If ATP is in excess, the enzyme
simply nicks the strand with Chi site. A Chi site or Chi
sequence is a short stretch of DNA in the genome of a
bacterium near which homologous recombination is
more likely to occur than on average across the
genome.
Unwinding continues and produces a 3’ ss-tail with
Chi near its terminus. This tail can be bound by RecA
protein, which promotes strand exchange with an
intact homologous DNA duplex.
RecBCD pathway of HR
where ATP is in excess.
When RecBCD reaches the end
of the DNA, all three subunits
disassemble and the enzyme
remains inactive for an hour or
more; a RecBCD molecule that
acted at Chi does not attack
another DNA molecule.
If Mg2+ ions are in excess, RecBCD cleaves both DNA
strands (by endonuclease). When RecBCD encounters a
Chi site on the 3' ended strand, unwinding pauses and
digestion of the 3' tail is reduced.

When RecBCD resumes unwinding, it now cleaves the

opposite strand (i.e., the 5' tail) and loads RecA protein
onto the 3’-ended strand. After completing reaction
on one DNA molecule, the enzyme quickly attacks a
second DNA, on which the same reactions occur as on
the first DNA.
Beginning of the
RecBCD pathway of
homologous
recombination where
Mg2+ is in excess
Under both reaction conditions, the 3' strand remains
intact downstream of Chi. The RecA protein is then
actively loaded onto the 3' tail by RecBCD.

At some undetermined point RecBCD dissociates from

the DNA, although RecBCD can unwind at least 60 kb of
DNA without falling off.
RecA initiates exchange of the DNA strand to which it is
bound with the identical, or nearly identical, strand in an
intact DNA duplex; this strand exchange generates a joint
DNA molecule, such as a D-loop which is cleaved to form
a Holliday junction.
The Holliday junction can be resolved into linear DNA
by the RuvABC complex or dissociated by the RecG
protein. Each of these events can generate intact
DNA with new combinations of genetic markers by
which the parental DNAs may differ. This process,
homologous recombination, completes the repair of
the double-stranded DNA break.

RuvABC is a complex of three proteins that mediate

branch migration and resolve the Holliday junction
created during homologous recombination in
bacteria. As such, RuvABC is critical to bacterial DNA
repair.