Rec A

1
Rec A

• and Related DNA Modifying Proteins
• Ryan Delahanty

2
Function of RecA

• Post replication repair
• Repairs dimers formed during replication or by
  exposure to UV-light or chemicals
• Part of the SOS response system in E. coli and
  other organisms
• Works with many other genes to repair mutations

3
The SOS Response

• RecA is part of the SOS response, a repair
  system.
• SOS response repairs mismatched strands of DNA.
• RecA triggers this response by some mechanism
  still unknown, but can do so in vitro in the
  presence of ATP and single stranded DNA.
• The response can be induced by UV crosslinking,
  drugs, thymine deprivation, or mutations of dna
  genes.

4
The SOS Response

• Response includes activation of long patch
  excision and Rec recombination repair pathways.
• uvr genes and recA genes function together.
• Repair systems work with and stimulate nuclease
  and polymerase systems.

5
Why DNA repair is important

• Double mutant uvr- and recA- cells can only
  tolerate about 2 dimers vs. 50 or more in wild
  type cells.
• Cell-cycle interference causing cessation of
  mitosis or cancer.

6
Symptoms of deficient DNA repair systems

• Some breast cancers caused by BRCA1 and uvr
• Xeroderma pigmentosum
• Ataxia-Telangiectasia
• Bloom Syndrome
• Cockayne Syndrome
• Fanconi Anemia

7
XP An Interesting Example
How frequently we accumulate mutations is
evident in people with xeroderma pigmentosum
8
How RecA Works

• LexA and recA are normally present at low levels
  in the cell. The lexA product inhibits lexA,
  recA, and many repair operons involved in the SOS
  response.
• When recA is stimulated, it causes autocatalytic
  cleavage of lexA.
• The genes involved in repair are now expressed as
  well as increased levels of lexA and recA.
• Some repair genes with 2 promoters express at
  higher levels when the lexA inhibitor is
  released.

9
How RecA Works

• The SOS box is the target of usual lexA binding
  and is found before all genes expressed in the
  SOS response. These include
• the expression of uvrA, B, C and D (dimer
  excision repair genes)
• recA is also responsible for homology searching
  and recombination repair
• ssb (protection of single stranded DNA)
• stiA (suppresses cell division)
• umuC and D (interact with DNA polymerase III to
  allow error prone replication past damaged DNA)
  and lexA is increased.
• Increase expression of lexA and recA.

10
How RecA Works
11
How RecA Works

• lexA cleavage increases the concentration of recA
  and lexA by about 50 fold.
• This ensures that the response to repair by recA
  is thorough and quickly halted by lexA when recA
  is no longer stimulated.

12
What I did

• Bioinformatic analysis of recA and comparison
  with other DNA repair/modifying proteins uvr,
  rad, topoisomerases, and other radiation
  activated proteins.

13
Sequence Selection

• Search for RecA proteins turned up 100
  sequences.
• uvr 55 sequences
• repair proteins 155 sequences
• rad 94 sequences
• redundant sequences and fragments were eliminated.

14
RecA Sequences The top 2 Sequences were listed
as RecA homologs. Fragments and repetetive
sequences have been removed. RA51_DROME
drosophila melanogaster (fruit fly). dna repair
protein rad51 homolog (reca protein homolog).
10/2000 RA51_MOUSE mus musculus (mouse). dna
repair protein rad51 homolog 1. 5/2000 REC1_MYXXA
myxococcus xanthus. reca protein 1.
5/2000 REC2_LACLA lactococcus lactis (subsp.
lactis) (streptococcus lactis). reca protein.
5/2000 REC2_MYXXA myxococcus xanthus. reca
protein 2. 5/2000 RECA_ACEPO acetobacter
polyoxogenes. reca protein. 5/2000 RECA_ACHLA
acholeplasma laidlawii. reca protein.
5/2000 RECA_ACICA acinetobacter calcoaceticus.
reca protein. 5/2000 RECA_ACIFA acidiphilium
facilis. reca protein. 5/2000 RECA_AERSA
aeromonas salmonicida. reca protein.
5/2000 RECA_AGRTU agrobacterium tumefaciens. reca
protein. 5/2000 RECA_ANAVA anabaena variabilis.
reca protein. 5/2000 RECA_AQUAE aquifex aeolicus.
reca protein. 5/2000 RECA_AQUPY aquifex
pyrophilus. reca protein. 5/2000 RECA_ARATH
arabidopsis thaliana (mouse-ear cress). dna
repair protein reca precursor. RECA_AZOVI
azotobacter vinelandii. reca protein.
10/2000 RECA_BACFR bacteroides fragilis. reca
protein. 5/2000 RECA_BACSU bacillus subtilis.
reca protein. 10/2000 RECA_BORBU borrelia
burgdorferi (lyme disease spirochete). reca
protein. 5/2000 RECA_BORPE bordetella pertussis.
reca protein. 5/2000 RECA_BRUAB brucella abortus.
reca protein. 5/2000 RECA_BURCE burkholderia
cepacia (pseudomonas cepacia). reca protein.
5/2000 RECA_CAMFE campylobacter fetus. reca
protein. 5/2000 RECA_CAMJE campylobacter jejuni.
reca protein. 10/2000 RECA_CHLAU chloroflexus
aurantiacus. reca protein. 5/2000 RECA_CHLPN
chlamydia pneumoniae (chlamydophila pneumoniae).
reca protein. 5/2000 RECA_CHLTE chlorobium
tepidum. reca protein. 5/2000 RECA_CHLTR
chlamydia trachomatis. reca protein.
5/2000 RECA_CHRVI chromatium vinosum. reca
protein. 5/2000 RECA_CLOPE clostridium
perfringens. reca protein. 5/2000 RECA_CORGL
corynebacterium glutamicum (brevibacterium
flavum). reca protein. 5/2000 RECA_CORPS
corynebacterium pseudotuberculosis. reca protein.
5/2000 RECA_DEIRA deinococcus radiodurans. reca
protein. 10/2000 RECA_ECOLI escherichia coli, and
shigella flexneri. reca protein.
5/2000 RECA_ENTAG enterobacter agglomerans. reca
protein. 5/2000 RECA_ERWCA erwinia carotovora.
reca protein. 5/2000 RECA_GLUOX gluconobacter
oxydans. reca protein. 5/2000 RECA_HAEIN
haemophilus influenzae. reca protein
(recombinase). 5/2000 RECA_HELPJ helicobacter
pylori j99 (campylobacter pylori j99). reca
protein. 10/2000 RECA_HELPY helicobacter pylori
(campylobacter pylori). reca protein.
5/2000 RECA_LACLA lactococcus lactis (subsp.
lactis) (streptococcus lactis). reca protein,
plasmid. RECA_LEGPN legionella pneumophila. reca
protein. 5/2000 RECA_LEPBI leptospira biflexa.
reca protein. 5/2000 RECA_LEPIN leptospira
interrogans. reca protein. 5/2000 RECA_MAGMG
magnetospirillum magnetotacticum (aquaspirillum
magnetotacticum). reca protein. 5/2000 RECA_METCL
methylomonas clara. reca protein.
5/2000 RECA_METFL methylobacillus flagellatum.
reca protein. 5/2000 RECA_MYCGE mycoplasma
genitalium. reca protein. 5/2000 RECA_MYCLE
mycobacterium leprae. reca protein.
5/2000 RECA_MYCMY mycoplasma mycoides. reca
protein. 5/2000 RECA_MYCPN mycoplasma pneumoniae.
reca protein. 5/2000 RECA_MYCPU mycoplasma
pulmonis. reca protein. 5/2000
RECA_MYCSM mycobacterium smegmatis. reca protein.
10/2000 RECA_MYCTU mycobacterium tuberculosis.
reca protein. 5/2000 RECA_NEIGO neisseria
gonorrhoeae. reca protein. 10/2000 RECA_NEIMA
neisseria meningitidis (serogroup a). reca
protein. 10/2000 RECA_NEIMB neisseria
meningitidis (serogroup b). reca protein.
10/2000 RECA_PARDE paracoccus denitrificans. reca
protein. 5/2000 RECA_PASMU pasteurella multocida.
reca protein. 5/2000 RECA_PORGI porphyromonas
gingivalis (bacteroides gingivalis). reca
protein. 5/2000 RECA_PRERU prevotella ruminicola
(bacteroides ruminicola). reca protein.
5/2000 RECA_PROMI proteus mirabilis. reca
protein. 5/2000 RECA_PROVU proteus vulgaris. reca
protein. 5/2000 RECA_PSEAE pseudomonas
aeruginosa. reca protein. 5/2000 RECA_PSEFL
pseudomonas fluorescens. reca protein.
5/2000 RECA_PSEPU pseudomonas putida. reca
protein. 5/2000 RECA_RHIET rhizobium etli. reca
protein. 10/2000 RECA_RHILV rhizobium
leguminosarum (biovar viciae). reca protein.
5/2000 RECA_RHIME rhizobium meliloti
(sinorhizobium meliloti). reca protein.
5/2000 RECA_RHOCA rhodobacter capsulatus
(rhodopseudomonas capsulata). reca protein.
5/2000 RECA_RHOSH rhodobacter sphaeroides
(rhodopseudomonas sphaeroides). reca protein.
5/2000 RECA_RHOVI rhodopseudomonas viridis. reca
protein. 5/2000 RECA_RICPR rickettsia prowazekii.
reca protein. 5/2000 RECA_SERMA serratia
marcescens. reca protein. 5/2000 RECA_SHISO
shigella sonnei. reca protein. 5/2000 RECA_SPIPL
spirulina platensis. reca protein.
5/2000 RECA_STAAU staphylococcus aureus. reca
protein. 5/2000 RECA_STRAM streptomyces
ambofaciens. reca protein. 5/2000 RECA_STRCO
streptomyces coelicolor. reca protein.
5/2000 RECA_STRLI streptomyces lividans. reca
protein. 5/2000 RECA_STRPA streptococcus
parasanguis. reca protein. 5/2000 RECA_STRPN
streptococcus pneumoniae. reca protein.
5/2000 RECA_STRPY streptococcus pyogenes. reca
protein. 5/2000 RECA_STRRM streptomyces rimosus.
reca protein. 5/2000 RECA_STRVL streptomyces
violaceus (streptomyces venezuelae). reca
protein. 5/2000 RECA_SYNP2 synechococcus sp.
(strain pcc 7002) (agmenellum quadruplicatum).
reca protein. RECA_SYNY3 synechocystis sp.
(strain pcc 6803). reca protein.
5/2000 RECA_THEAQ thermus aquaticus. reca
protein. 5/2000 RECA_THEMA thermotoga maritima.
reca protein. 5/2000 RECA_THETH thermus aquaticus
(subsp. thermophilus). reca protein.
5/2000 RECA_THIFE thiobacillus ferrooxidans. reca
protein. 5/2000 RECA_TREPA treponema pallidum.
reca protein. 5/2000 RECA_VIBAN vibrio
anguillarum. reca protein. 5/2000 RECA_VIBCH
vibrio cholerae. reca protein. 10/2000 RECA_XANCI
xanthomonas campestris (pv. citri). reca protein.
5/2000 RECA_XANCP xanthomonas campestris (pv.
campestris). reca protein. 5/2000 RECA_XANOR
xanthomonas oryzae. reca protein.
5/2000 RECA_XENBV xenorhabdus bovienii. reca
protein. 5/2000 RECA_YERPE yersinia pestis. reca
protein. 5/2000
15
Sequence characteristics

• 99 Sequences Total
• Average Protein Length 360 amino acids
• Anomalies
• RecA_Mycle and RecA_myctu mycobacterium leprae
  (711AA) and mycobacterium tuberculosis (790AA)
  are both self splicing proteins that involve a
  post-translational excision of an intervening
  region (intein) followed by peptide ligation.
• RecA_Arath found a slightly longer (439AA) recA
  protein in arabidopsis thaliana
• RecA_Lacla recA on a phage resistance plasmid in
  lactococcus lactis
• Average molecular weight of proteins 45000
  daltons
• no introns
• SOS box operators
• proteins are soluble in cytosol

16
Protein Homology

• 12 recA sequences and 12 ra51 sequences

17
Protein Homology
18
Protein Homology
RecA Consensus (100) Consensus

Consensus
---------- ----------
Consensus ---------- ----------
---------- A----G-P-- ---E--G-ES
Consensus -GKT....T- --------Q- ----..----
D--------. ...-----G- Consensus
-....---L- ---...---G E--------- ----------
-DSV--L-P- Consensus -E--G-----
-----A---S ---------- --------F- -Q--------
Consensus ---------- ----------
--------.. ...------- ----------
Consensus ---------- ---------- ----------
---------- ---------- Consensus
---------- ---------- ---------- ----------
---------- Consensus ----------
---------
Consensus

Consensus
Consensus

Consensus

Consensus

Consensus
Consensus

Consensus

Consensus

19
Protein Homology
RecA Consensus (95) Consensus
---------- ---------- ---------- ----------
---------- Consensus ----------
---------- ---------- ---------- ----------
Consensus ---------- ----------
---G----D- A-G-GG-P-G R--E--G-ES
Consensus -GKT----T- -----A--Q- -G--------
DAE---D--- ---YA---G- Consensus
--------L- ------P--G E--L------ ----------
-DSVAAL-P- Consensus -E--G-----
--G--AR-MS ---R------ --------F- NQ-R-K-G--
Consensus ------E--- -T-GG-ALKF
------R--- -----R---- ----------
Consensus ---------- --------K- K-------P-
---------- --G------- Consensus
---------- ---------- ---G------ ----------
---------G Consensus ----------
---------- ---------- ---------- ----------
Consensus ---------- ----------
---------- ---------- ----------
Consensus ---------- ---------- ----------
---------- ---------- Consensus
---------- ---------- ---------- ----------
---------- Consensus ----------
---------- ---------- ---------- ----------
Consensus ---------- ----------
---------- ---------- ----------
Consensus ---------- ---------- ----------
---------- ---------- Consensus
---------- ---------- ---------- ----------
---------- Consensus ----------
---------- ---------- ---------- ----------
Consensus ---------- ----------
--------
20
Protein Homology
Rad23 Consensus (95) Consensus
-----T-K-- ---------- P--T----K- K---------
------KLIY Consensus -GK-L-D---
---------- -VV-MV---K ---------- ----------
Consensus ---------- ------P---
-T------T- --P------- -P--------
Consensus --P-E----T ---T------ ----------
---------V -G-------- Consensus
-IM-MGY-RE -V--ALRA-- NNP-RAVEYL L-GIP-----
-E-------- Consensus ----------
---------- ---A------ ---------- -----G--P-
Consensus ---------- --RQ----NP
--L--LL--- ----PQL--- I----E-F--
Consensus ML-E------ ---------- ---------G
---------- ---------- Consensus
TP----AI-R L--LGF---L VIQ-YFAC-K NE--AAN-L-
----D--
21
Protein Homology
Ra51 and RecA Consensus (100) Consensus
---------- ---------- ---------- ----------
---------- Consensus ----------
---------- ---------- ---------- ----------
Consensus ---------- ----------
---------- ---------- ----------
Consensus ---------- ---------G ----------
-G---G---E --G----GK- Consensus
----T----- ---------- -----ID-E- ----------
---------- Consensus ----------
---------- ---------- -------DS- --L-------
Consensus G--------- -A--------
---------- --------NQ ----------
Consensus -F-------- GG-------- -R--------
---------- ---------- Consensus
---------- ---------- ---------- ----------
---------- Consensus ----------
---------- ---------- ---------- ----------
Consensus ---------- ----------
---------- ---------- ----------
Consensus --
22
Protein Homology
r51, r23 and RecA Consensus (50)
Consensus ---------- ---------- ----------
---------- ---------- Consensus
---------- ---------- ---------- ----------
---------- Consensus ----------
---------- ---------- ---------- ------G---
Consensus ---------- ----------
---------L D--L-GGI-- ---TE--G--
Consensus --GKT--CH- -A-------- -------A-Y
IDTE------ R--------- Consensus
---------- ---------- ---------- ----------
--L------- Consensus -----S----
L--VD----- -R-------- G--------- L------L--
Consensus -A------V- -TNQ------
G--------- --P-G----- ----------
Consensus ---GN--A-- ---------- --G--R----
-D-----E-- ---F-I---G Consensus
---------- ---------- ---------- ----------
---------- Consensus ----------
---------- ---------- ---------- ----------
Consensus --
23
Protein Homology
r51, r23 and RecA Consensus (90)
Consensus ---------- ---------- ----------
---------- ---------- Consensus
---------- ---------- ---------- ----------
---------- Consensus ----------
---------- ---------- -----A---- ---F------
Consensus ---------- ---------G
S--LD--L-- GG---G-I-E --G----GKT
Consensus ----T-A--- -------GG- -----ID-E-
---P------ A---G----- Consensus
---------- ---D-----L ---------- ---L--VDS-
-AL------- Consensus G--------L
-AR-M----- -R-L------ --------NQ ----------
Consensus -F-------- GG--------
-R---R---- ---------- ----------
Consensus ---------- ---------- ----------
---------- ---------- Consensus
---------- ---------- ---------- ----------
---------- Consensus ----------
---------- ---------- ---------- ----------
Consensus --
24
(No Transcript)
25
(No Transcript)
26
Cladogram of RA51 and Rec A relationships
27
Images of recA Bound to DNA
28
(No Transcript)
29
Computer-enhanced Image of Bound recA
30
Motifs

• Found many ATP/GTPase binding domains among
  proteins.
• The best conserved of these motifs is a
  glycine-rich region, which typically forms a
  flexible loop between a beta-strand and an
  alpha-helix. This loop interacts with one of the
  phosphate groups of the nucleotide.
• -Consensus pattern AG-x(4)-G-K-ST

31
Conclusion

• RecA has a high degree of homology to RA_51 and
  RA_23 which indicates that these proteins are
  likely eukaryotic homlogs of the original RecA
  repair protein. They show structural similarity
  in their profiles, sequences, motifs, and
  crystalized structures. -fin.