This document summarizes research on homologous recombination pathways and their regulation. It describes the key stages of homologous recombination, including DNA end resection, formation of the presynaptic filament by Rad51/Dmc1, synaptic complex assembly, and D-loop formation. It also discusses several factors that modulate homologous recombination by facilitating presynaptic filament assembly, synaptic complex formation, D-loop migration, and favoring crossover or non-crossover outcomes. The Yale laboratories aim to further elucidate the mechanisms and regulation of homologous recombination and its role in genome maintenance.

1. INVESTIGATIONS OF HOMOLOGOUS
RECOMBINATION PATHWAYS AND
THEIR REGULATION
JOURNAL NAME:- YALE
JOURNAL OF BIOLOGY AND
MEDICINE (2013)
VOL. NO. 86
PAGE NO. 453-461
PRESENTED BY:- VIKAS
M.Sc. ZOOLOGY

2. CONTENTS
Introduction
Overview
Process of HR
Conclusion
Future prospectives
References

3. INTRODUTION
 DNA is continually damaged by many endogenous and
exogenous agents.
 Charles Radding and Paul Howard-Flanders of Yale has led to
insights regarding HR mechanism in Bacteria and also provide
experimental frameworks to guide similar studies in
eukaryotes.
 Radding contributed to the idea that recombination begins
with the invasion of a homologous duplex by ssDNA.
 They elucidate the biochemical mechanism by which the
RecA recombinase promotes the homologous DNA pairing
and strand exchange reaction that underlies the HR mediated
process.

4. OVERVIEW
 The process begins with the DNA end resection, in which
nucleolytic degradation of 5' strands takes place and leaves a
3' ssDNA overhang.
 This ssDNA is then coated by replication protein A(RPA),
which is displaced by a recombinase to yield the presynaptic
filament.
 The synaptic complex formed when filament pairs with
homologous dsDNA.
 The invasions of the homologous duplex, forming a structure
called Displacement loop or D-loop.
CONT…

5. OVERVIEW
 After DNA synthesis extends
the D-loop structure can be
dismantled and leads to a non
crossover products.
 Alternatively, a double holiday
junction can form, which can
be resolved by specialized
nucleases.
 These junction can also be
dissolved by a helicase-
topoisomerase complex to
yield a non-crossover products.

6. DNA END RESECTION
 To recruit the proteins that
catalyze DSB repair, 3' DNA
tails first be created at break
site.
 This resection process takes
place with the help of protein
complex MRX/MRN which
possesses 3' to 5' exonuclease
and structure-specific
endonucleases.
 Long range resection is
catalyzed by either the 5' to 3'
exonuclease Exo1 or ssDNA
endonuclease Dna2.

7. EUKARYOTIC RECOMBINASE: Rad51
AND Dmc1
 The ssDNA is first engaged by RPA, which is then replaced by a
recombinase, either Rad51 or Dmc1 to mediate Homologous
DNA pairing.
 Rad51 and Dmc1 have key properties of the recombinase
protein filament.
 Within the presynaptic filament DNA is stretched. This
extended DNA is characteristic of a catalytically active
presynaptic filament.
 Than in the homologous pairing rexn, the presynaptic filament
engages the duplex DNA molecule and forms synaptic complex
 Finally, invasion of the duplex by presynaptic filament yields a
DNA joint called D-loop.

8. PROMOTION OF PRESYNAPTIC FILAMENT
ASSEMBLED BY RECOMBINATION MEDIATORS
 The most well-studied HR mediators
are Rad52 (S. cerevisiae) and BRCA2
(human).
 BRCA2 is essential for HR and
maintenance of genetic stability in
mammalian cells, binds with DNA
and interact with RAD51.
 It is shown that a polypeptide
derived from BRCA2 containing the
BRC repeats 3 and 4 and DNA binding
domain possesses recombination
mediator activity.

9. HR FACTORS THAT FACILITATE SYNAPTIC
COMPLEX ASSEMBLY
 Several HR factors, namely RAD51AP1,
the tumor suppressor PALB2, and the
Hop2-Mnd1 complex enhance the
efficiency of synaptic complex
assembly.
 Both RAD51AP1 and PALB2 bind DNA
and can individually, co-operate with
the RAD51 filament to capture duplex
DNA and assemble the synaptic
complex.
 In Yeasts, the Hop2 and Mnd1 proteins
forms a heterodimeric complex to
promote crossover by enhancement of
Dmc1-mediated Homologous DNA
pairing.
 The mammalian Hop2-Mnd1 complex
can functions with both RAD51 and
Dmc1.

10. ROLE OF Rad54 AND Rdh54 IN
HOMOLOGOUS DNA PAIRING
 Rad54 and Rdh54 interact with Rad51 and Dmc1 and
enhance the homologous DNA pairing reaction.
 When translocating on DNA, Rad54 and Rdh54 generate
negative supercoiling that induces transient separation
of DNA strands.
 Both protein possesses a chromatin remodelling activity
that enables D-loop formation.
 These protein also mediate the migration of nascent
Holiday structure made during HR and remove Rad51
and Dmc1 from dsDNA.

11. PROMOTION OF THE NON-CROSSOVER
SDSA PATHWAY BY THE Mph1 HELICASE
 In S. cerevisiae, the Mph1 helicase
is a major negative regulator of
crossover HR.
 It acts by resolving D-loop
intermediates via the non-
crossover pathway of SDSA.
 Specifically, Mph1 utilizes its
helicase function to dissociate the
invading strand from the D-loop
structure.
 In fission yeast and human
orthologs of Mph1 are found viz.
Fm11 and FANCM
respectively.They also have similar
enzymatic activities like Mph1.

12. CROSSOVER SUPPRESSION VIA DOUBLE
HOLIDAY JUNCTION DISSOLUTION BY BLM-
TOPO IIIα-RMI1-RMI2
 The double Holiday Junction
dissociation reaction is
mediated by the BLM helicase in
conjunction with type1A
topoisomerase TopoIIIα.
 RPA and the OB-fold containing
RMI1-RMI2 complex associate
with BLM-TopIIIα and enhance
the dHJ dissolution reaction.

13. Srs2 AND REQ5:NEGATIVE HR REGULATORS
THAT DISASSEMBLE THE PRESYNAPTIC
FILAMENT
 In budding yeast Srs2 helicase helps in supression of
spurious HR events via disruption of Rad51 presynaptic
filament.
 This process is stimulated by RPA and also requires
complex formation between Srs2 and Rad51.
 In human RecQ family helicase RECQ5 helps in
supression of spurious HR events.
 RECQ5 interacts with RAD51 and inhibits presynaptic
filament assembly.

14. CONCLUSION
 Their studies have provided insights into how
the HR machinery forms and subsequently
processes DNA joints during recombination.
 They also contributed to the regulatory
mechanism that influence the frequency and
outcome of HR.

15. FUTURE PROSPECTIVES
The YALE Laboratories will continue
to help to elucidate the mechanism
and regulation of HR and its role in
genome maintenance.

16. REFERENCES
 Trujillo KM, Yuan SS, Lee EY, Sung P. Nuclease activities
in a complex of human recombination and DNA repair
factors Rad50, Mre11, and p95. J Bio1 Chem.
1998;273(34):21447-50.
 San Filippo J, Sung P, Klein H. Mechanism of eukaryotic
homologous recombination. Annu Rev Biochem.
2008;77:229-57.
 Raynard S, Niu H. Sung P. DNA double-strand break
processing:the beginning of the end. Genes Dev.
2008;22(21|):13861-4