GENETIC RECOMBINATION
1. Distinguish between homologous recombination (general recombination) and sitespecific recombination.
Regarding homologous recombination:
2. Describe the process strand invasion and its catalysis by RecA protein.
3. Describe the key steps in double strand break repair by homologous recombination.
4. Describe the key steps in homologous recombination during meiosis and how
formation and resolution of a Holliday junction can lead to crossover between
homologous chromosomes.
5. Understand how gene conversion can occur as a consequence of homologous
recombination.
6. Understand the consequences of inappropriate homologous recombination between
closely related repetitive elements. Describe how cells prevent this from occurring.
Regarding site-specific recombination:
7. Describe the mechanism of transposition utilized by DNA-only transposons.
8. Describe the mechanisms of transposition utilized by retroviruses and retroviral-like
retrotransposons.
9. Describe the mechanisms of transposition utilized by nonretroviral retrotransposons.
GENETIC RECOMBINATION
1. Homologous recombination
a. Exchange between pair of DNA segments that are homologous (identical or
nearly identical in sequence)
b. Important for exchanging segments between maternal and paternal chromosomes
during meiosis and in DNA repair, especially breaks in replication forks and other
double strand breaks
2. Strand Invasion
a. Occurs between two DNA duplexes in segments of homology
b. Single stranded region generated in one duplex by various mechanisms (see
below)
c. Strand invasion, where single strand from one duplex base pairs with
complementary strand from homologous duplex, displacing other strand from
homologous duplex
d. Results in heteroduplex- region where each strand of DNA that is paired was
originally present in two different molecules
e. Branch point (where stand exchange occurs) can migrate in one direction,
extending heteroduplex region
3. RecA protein catalyzes strand invasion
a. Associates invading single strand with DNA duplex being invaded
b. Creates three-stranded intermediate in which bases transiently flip out from
double helix
c. Results in heteroduplex formation- stable pairing of original single strand with
complementary strand from double helix, other strand from double helix is
displaced
d. RecA is DNA-dependent ATPase; associates more tightly with DNA in ATPbound form
e. ATP-bound RecA preferentially added to one end of RecA filament; ATP
subsequently hydrolyzed; heteroduplex extended in one direction
f. Brca1 and Brca2 are accessory proteins for a type of RecA protein that is
important for repairing broken replication forks
4. Double strand break repair by homologous recombination
a. Preferred method shortly after DNA replication when associated sister chromatids
are attached
b. Exonuclease degrades 5’ ends in broken chromosome
c. Strand invasion by protruding 3’ end into homologous DNA duplex
d. Branch point migration coupled with DNA synthesis at broken strand, using
strand of duplex being invaded as template
5. Meiotic recombination
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a. Recombination for meiosis or for DNA repair involves flexible set of reactions
involving strand invasion, DNA synthesis, and branch point migration that can
vary to produce different outcomes
b. Occurs between homologous maternal and paternal chromosomes
c. Nuclease makes double strand break
d. Exonuclease degrades 5’ ends in broken chromosome
e. Stand invasions and branch migration lead to formation of Holliday junction,
which is intermediate structure where four strands come together and cross;
double Holliday junction usually forms
f. Multiple structures of Holliday junction due to isomerization
g. Resolution of Holliday junction occurs by cutting stands and regenerating two
separate DNA duplexes
h. Depending on how resolution proceeds, can regenerate original DNA duplexes
that contain heteroduplex region or can generate crossover in which segments of
each chromosome are swapped; crossover events are rare and average 2-3 per
chromosome
6. Gene conversion
a. For small segments of chromosome, divergence from standard distribution of
allele to haploid progeny cells during meiosis in which three receive one allele
(version of gene) while one receives other allele
b. Occurs at regions produced during homologous recombination
 limited DNA synthesis following strand invasion produces segments in which
three strands contain one allele (heteroduplex in one of the duplexes),
 heteroduplex region at crossover site
c. mismatch repair at heteroduplex can result in both stands having same allele while
other allele is lost
7. Preventing inappropriate homologous recombination
a. Recombination between closely related repetitive elements can lead to deletions
in genome
b. Mismatch repair system has second function that detects strand exchanges
between similar (but not identical) elements and aborts the process
8. Site-specific recombination
a. Movement of mobile genetic elements between nonhomologous sites in the
genome; elements range in length from 1000 to about 12,000 nucleotide basepairs
b. Mobile elements typically encode enzymes that catalyze their mobilization;
specific short DNA sequences at both ends of element are usually required
c. Human genome abundant in these elements; most are mutated and thus are no
longer able to mobilize; occasional mobilization can lead to mutations
9. Transposons (transposable elements)- capable of mobilizing to random sites
a. DNA-only transposons
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Same short sequence at each end of transposon that is recognized by
transposase
Cut-and-paste transposition: transposase molecule binds to end sites;
dimerization of transposase leads to DNA loop; transposase cuts both ends of
loop to release transposon; inserts into new random site
Replicative transposition: variation in which transposon is first replicated;
copy is moved while original remains
b. Retroviral-like retrotransposons and retroviruses
 Retroviruses have a protein capsid packed with a single-stranded RNA
genome that encodes reverse transcriptase (a DNA polymerase that uses an
RNA template), integrase (a transposase), and viral coat proteins
 Following retroviral infection, reverse transcriptase produces double-stranded
DNA from RNA genome; integrase recognizes specific sequence at ends of
DNA and catalyzes insertion into random site
 Cellular enzymes transcribe integrated DNA to produce new virus particles
 Retroviral-like retrotransposons use the same mechanism for transposition but
do not encode proteins for a functional viral coat; following transcription of a
retrotransposon, the RNA is reverse transcribed into double-stranded DNA,
and integrated to a new site within same cell
c. Nonretroviral retrotransposons
 Transpose via an RNA intermediate that is reverse transcribed to DNA;
mechanism of transposition is distinct from retroviral-like retrotransposons
 Complex containing endonuclease, reverse transcriptase, and transposon RNA
nicks target DNA at point of insertion; generates free 3’-OH in target DNA
that serves as primer for replicating first strand of transposon, which remains
attached to target DNA; subsequent steps synthesize second strand
 LINES, SINES comprise large proportion of human genome; most copies
have mutated and are no longer mobile, but some can mobilize infrequently to
generate new mutations
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