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Histone-Catalysed DNA Lesion Excision
1. Rapid Histone –Catalyzed
DNA Lesion Excision and
Accompanying Protein
Modification in Nucleosomes
and Nucleosome Core
Particles
Diksha Jain
Karpagam Sudhakar
2. Introduction
• The initial step of BER involves enzymatic
activities of DNA glycosylases that process the N-
glycosylic bonds linking the target bases and their
deoxyribose sugars
• The central intermediate is an abasic [apurinic ⁄
apyrimidinic (AP)] site in DNA
• In a given day, each cell produces between 10,000
and 50,000 AP lesions
• Oxidized abasic sites eg. C4-AP, L, DOB arise
following hydrogen atom abstraction from the 2'-
deoxyribose ring of a nucleotide
3.
4.
5. • Produced by γ-radiolysis , cytotoxic antitumor
agents, such as bleomycin and the enediynes (e.g.
neocarzinostatin)
• AP, C4- AP, and DOB yield DNA interstrand
cross-links, A number of cytotoxic
pharmacological agents, such as nitrogen
mustards and mitomycin C
• C4-AP, DOB, and L inactivate base excision repair
enzymes
• Histone proteins also catalyze excision of the
electrophilic lesions, AP, C4-AP, DOB and L
6.
7.
8.
9. • Chromatin is composed of monomeric
nucleosome units. In each nucleosome DNA (~145
bp) wraps around an octameric core of 4 different
highly positively charged histone proteins H2A,
H2B, H3 and H4
• The histones also contain lysine-rich termini
(“tails”) that protrude through the nucleosome
core particle (NCP)
• Post-translational modification of the lysines in
the conformationally mobile tails plays an
important role in gene regulation
• Histone tails have been shown to catalyze the
excision of AP sites almost 500 fold compared to
free DNA
10.
11.
12.
13. DOB
• Minor product of DNA oxidation resulting from
H atom abstraction from C5` of DNA sugar
backbone
• Formed at the 5` terminus concomitantly with a
strand break
• Undergoes β-elimination
• Forms Interstrand crosslinks (ICLs)
• Also, irreversibly inhibits DNA Pol β and λ
14. • We are going to investigate the
reactivity of DOB at specific sites
within NCPs and Nucleosomes
15. Design and Preparation of NCPs and
Nucleosomes containing DOB
• Widom 601 strong positioning sequences were
used to bind to histone core because of strong
affinity
• They have the most flexible dinucleotide type, TA
at minor groove-inward positions where DNA
distortion is energetically most challenging and
G|C-rich elements, which are predisposed to
major groove compression, at major groove-
inward positions
• Independent lesions were to be generated at
specific sites SHL 1.5 (DOB89), SHL 4.5 (DOB119),
and at SHL 0 (DOB 73)
16.
17. • Oligonucleotides containing DOB precursor at
specific sites were generated
• Complementary strands were also created
• Hybridization of the two strands, gave the ternary
complex
• Internal radiolabeling and restriction enzymes
site were incorporated at <17 bp from the DOB
lesion, to facilitate separating DNA strands
containing DOB from those without it
• Reconstitution was done with Histones
18. DOB reactivity in NCPs
• Reconstituted nucleosomes were used
• Nucleosomes containing DOB precursor were
photolyzed at 350 nm, for 5 min at room
temperature
• Aliquots were removed at appropriate times
• After phenol extraction, restriction digestion was
done (AccI for position 89, MseI for position 73,
MspI for position 119, MluCI for position 159)
• PAGE was performed
19.
20.
21. • Separate experiments were performed for DOB
reactivity in NCPs and in free DNA
• The half-life of DOB in NCPs depended upon its
position, but was greatly reduced in general
compared to free DNA
• The translational position has little effect on the
DOB half-life, which vary only ∼2-fold.
• The variation of DOB reactivity between these
four substrates is small compared to the overall
acceleration observed in NCPs (∼275−1500-fold),
suggesting that the presence of the lesion in the
NCP is more important than its rotational
orientation.
22.
23. DNA-Protein Cross-links
• Reconstituted nucleosomes containing DOB
precursor were photolyzed at 350 nm for 5 min at
r.t. and immediately incubated at 37 °C for the
duration of the time course experiment.
• Aliquots were removed at appropriate times
• SDS PAGE was performed
24.
25.
26. • The intermediacy of DPCs in DOB excision was
probed further using NaBH3CN, which reduces
Schiff-bases to alkylamines.
• On account of rapid DOB reactivity, NCPs were
photolyzed (5 min) in the presence of NaBH3CN
• SDS PAGE was performed
27.
28. Identification of Histone
involved in DPC formation
• The DPCs formed from DOB in the presence of
NaBH3CN were separated by SDS PAGE and the
histone proteins cross-linked to DOB were subjected to
mass spectrometry analysis following in-gel protease
digestion
• The identities of cross-linked proteins were confirmed
by comparing the mass spectra with those obtained
from wild-type (WT) histone proteins.
• DOB119 - one major (H2A) and two minor bands were
observed
• DOB73 – yield multiple bands, unsuccessful attempt
29.
30. Role of H4 Lysine residue in Catalyzing
DOB89 reactivity
• Sunstituting 5 Lys residue in the H4 tail with
arginines, overall charge is reatained but basicity
and nucleophilicity is reduced
• But 50 fold increase in acceleration is unaccounted for
31. FATE OF DOB89
• Four histone proteins were purified by HPLC
following incubation of NCP with DOB, characterized
by MS
• Only Modifications in H4 were detected – 66 and 84Da
additional ions
38. • Also, reactivity at Dyad position was studied. No
terminal tail from a histone protein is in close
proximity to DOB73, but cross-linking
experiments using an electrophilic probe revealed
interactions between the dyad position and
histones H2A, H3, and H4
• Such interactions may depend on the presence of
the linker region
• In the presence of the linker DNA, DOB73
decomposed at a slightly higher rate than in the
corresponding NCP, making it ∼500-fold faster as
compared to that in the free DNA
• No interactions between linker DNA and the
histone proteins.
39. Reactivity of DOB within Linker
DNA
• DOB was incorporated at 159 and 176
• Slow decomposition as compared to DOB73, 89
and 119 but still faster relative to free DNA
• Indicates Histone tails has some contribution
• The higher rate of excision at DOB159 than
DOB176 is can also be attributed to histone
protein participation
POSITION DISTANCE
FROM CORE
HALF LIFE IN
THE LINKER
HALF LIFE IN
FREE DNA
DECREASE IN
REACTIVITY
IN FREE DNA
159 13 bp 44.4 ± 4.8 min 99.6 ± 4.1hr 135 fold
176 30 bp 190.8 ± 12.6 min 147.1 ± 7.5 h 46 fold
41. • Histone H3 was separated from other histones in SDS
PAGE – Lys C and trypsin digest
• Peptide fragment 1-9 in modified H3 but not in wild
type H3 and the difference was 66 Da – Lys4 is
modified
• LC MS/MS 3-8 fragment – was 66 Da higher than
unmodified
• Total length of N-terminal H3 tail is expected to
be 113Å
• DOB176 is expected to be 105Å away, so only
most distal amino acids are expected to interact
• DOB159 is only 47.6Å away, so Lys 4, 9, 14, and 18
could potentially interact
42. SIGNIFICANCE OF H3K4
• H3K4- Trimethylation modulate proper transcription
• Acts as binding site for HDAC to induce closed
conformation of chromatin
• Methylated H3K4 also associates with acetylases,
indicating a role in transcriptional activation
• The pyrrolone modification does not resemble any
methylated form of lysine in terms of charge or shape
• Thus, modification at this residue can adversely affect
cell signalling
43. Other factors
• ~50 fold accelerated reactivity of DOB89 is
accounted in K5,8,12,16,20R/H18A H4
• DOB176 undergoes β-elimination 46-fold faster in
the nucleosome than in the corresponding free
DNA. No evidence for direct histone interactions
with this position in the linker DNA
• Some other factors are responsible for this
• Hypothesis- random interactions between unbound
Histone in solution could be responsible
44. • Excess carrier DNA was added – to remove any free
histone
• In complementary study, Nucleosome solution was
diluted to weaken any internucleosomal interaction
• The reactivity of DOB176 showed~3 fold decrease
• Interaction between free histone octomers and
nucleosome DNA are responsible for a small portion
POSITION WITHOUT
ADDITION OF
CARRIER
DNA
1 FOLD
EXCESS
2 FOLD
EXCESS
DOB at 176 3.2 ±0.2 h 4.1 ± 0.1 h 4.5 ± 0.2 h
45. CONCLUSION
• DOB is a lesion produced by exogenous DNA
damaging reagents including ionizing radiation and
anticancer antibiotics
• Highly reactive in NCP (as much as 1,500 fold)
• DOB competes with the repair enzymes Pol β and Polλ
• Searching for Pyrrolone at lysine can be used to detect
intracellular DOB production
• The pyrrolone structure disrupts the homeostasis –
mimics acetylation and affects binding of proteins
• The lesions in linker region also plays significant role
Base excision repair is the most important cellular defense mechanism that evolved to avert the deleterious effects of the damaged bases in DNA. It has been estimated that AP site formation through the spontaneous hydrolytic loss of purines generates some 10 000 AP sites per day in a mammalian cell. Rest is the work of DNA glycolsylases.
2-deoxyribonolactone (dL) which is generated by initial hydrogen abstraction from the deoxyribose C1 carbon, followed by O2 addition and base loss
C4′-oxidized abasic site (C4-AP), 2-Deoxyribonolactone (L), 5′-(1,4-dioxobutane) (DOB)
Some of these cross-links are misrepaired by bacterial nucleotide excision repair in vitro and converted into double strand breaks.
What I am trying to explain here is, these AP sites are naturally formed as an intermediate in the repair pathway, and also induced by various agents. Now, if they get repaired, they are not an issue. But when they are not repaired, they are mutagenic.
Activated Fe·BLM abstracts a H atom from the C-4′position of deoxyribose at the primary site (typically 5′-G-Py-B-3′ of one strand of the hairpin DNA, where B = any nucleobase), producing an AP (apyrimidinic/apurinic) site (I)
AP forms ICLs selectively with the dG in 5'-C-AP , sequences under mild reducing conditions (NaBH3CN)
the presence of a 1,4-dicarbonyl functional group in DOB, which reacts rapidly with primary amines dramatically alter the outcomes of interactions. Covalent modification of Lys84 (9) and indirect evidence for reaction at Lys72. Both lysines have been implicated in Schiff base formation during 5'-dRP excision
Now, the difference between a NCP and Nucleosome is that nucleosome has 1 linker DNA attached
These tails are subjected to various modifications such as acetylation, methylation, phosphorylation, ubiquitination etc. This either creates sites for the recruitment of specific factors or modify existing sites so as to abolish previous interactions and thus alter the expression states.
Acetylation (ac) occurs exclusively on lysine residues of histone peptides by ‘Histone acetyltrasferases‘ (HAT). Acetylation causes neutralisation of the positively charged lysine residues, hampering the histone-DNA interaction. Histone acetylation is predominantly associated with transcriptional activation.
Crystal structure of the nucleosome core particle at 2.8 Å resolution.
5′-(2-phosphoryl-1,4-dioxobutane) (DOB)
Alignment of the highest affinity sequences derived by SELEX allowed Widom and colleagues to predict a consensus sequence for maximum affinity histone octamer binding over the central 73 bp of the nucleosome, this approach apparently did not allow full optimization of the outer H2A–H2B binding sites. Oligonucleotides were synthesized on an Applied Biosystems Incorporated 394 oligonucleotide synthesizer
Structure of one half of the NCP, as we can see there is only one round on DNA around the core. The position of each base pair with respect to the histone
octameric core can be referred by its superhelical location (SHL), it is a translational parameter, relative to the dyad position (SHL 0), where the twofold axis of the octamer is. Each turn of the duplex corresponds to one unit change in the SHL. Herein, we describe the histone-catalyzed excision of DOB in nucleosome core particles (NCPs) and nucleosomes. Modified nucleotides are shown as spheres
The DNA at SHL 1.5 is bent, is a preferred binding site for DNA-damaging reagents and is close to the lysine-rich N-terminal tail of histone H4. DNA
kinking in the region around SHL 4.5 severely disrupts base stacking.4 SHL 4.5 is also in close proximity to the N-terminal tails from histone H2A and H2B. The DNA is held more tightly at the dyad region (SHL 0), but is not as closely positioned to any N-terminal histone tails as at SHL 1.5 and 4.5.
Modified nucleotides are shown as spheres
SO it means, the
DOB was excised by the Histone tails, and the reactivity was measured. quenched with NaBH4.
Reactivity of DOB monitored as a function of time by 20% denaturing PAGE in NCPs containing DOB89 (A), DOB73 (B), DOB119 (C), and DOB89* (D). *indicates the different flanking sequence of DOB89. DOB excision from nucleosomal DNA exhibited first-order kinetics, here we are plotting only the conc of DOB, not log of that.
A previous study on AP reactivity in NCPs indicated that the effect of rotational position was modest (<3-fold)
the three positions investigated, DOB at SHL 1.5 (DOB89) exhibited the shortest half-life of 7−8.5 min, depending upon the flanking sequence, indicating that the latter has little effect on the lesion’s reactivity in NCPs. The rates of disappearance of DOB at SHL 4.5 (DOB119) and DOB73 (SHL 0) are only approximately 2-fold slower than DOB89.
The maximum DPC yield was ∼30% for DOB89 and DOB73, and ∼20% for DOB119. The maximum DPC yields were reached during the first half-life of the reactions (<5 min), and decreased rapidly thereafter. DPC formation is proposed to result from Schiff-base formation between the lesion and lysine residues in the histone proteins.
The DPCs were formed in slightly higher yields (40−50%) compared to when the reducing agent was absent, and the yields remained relatively unchanged over time for DOB89 and DOB119 (Figure 4B). DOB73 produced DPCs in the highest yield (>60%) among the three DOB sites, but after reaching a maximum at 5 min they gradually decreased to a comparable level as at the other sites. The decomposition of reduced DPCs formed at DOB73 could be attributed to the β- elimination from 4. DOB lesions are excised completely within 1 h, the relatively low DPC yield (40−60%) suggests that trapping by NaBH3CN was incomplete and/or that Schiff-base formation is not the sole mechanism for DOB excision.
MALDI-TOF mass spectra of peptide fragments obtained from in-gel acetylation and trypsin digest of (A) WT histone H4 and (B) the DPC produced by DOB89. (The range of amino acids corresponding to each peptide is in parentheses.) (C) Peptide sequences and respective calculated m/z. indicated that DOB89 mainly cross-linked with this protein. You can also see in the structure, that h4 is ic close proximity to DOB89
Histone Variants were created. arg pka -12.5 and lys -10.5. Five Lys (5,8,12,16,20) residues to Arg – 8 fold increase in half life
K(5,8,12,16,20) R and His 18 to Ala – 15 fold increase in half life.
To test this proposal, the four histone proteins were purified by HPLC following incubation of NCP containing DOB89 and characterized by mass spectrometry. Consistent with DPC formation (Figure 5), only modification of histone H4 was detected. This protein contained two additional ions, which were 66 and 84 Da greater than the unmodified protein
To achieve higher resolution, the purified H4 obtained from the NCP containing DOB89 was subjected to protease digestion. MALDI-TOF MS analysis
following thermolysin digestion, which cleaves proteins at the amino termini of hydrophobic residues, revealed that the 20- amino acid N-terminal tail was the only region modified. Two fragments (amino acids 1−9 and 10−20) whose masses were increased by 66 Da compared to the unmodified ones were
observed
Thermolysin cleaves at N terminus of hydrophobic residues leucine, phenylalanine, valine, isoleucine, alanine and methionine. SGRGK5GGK8GLGK12GGAK16*RHRK20VLRDNIQGITKPAIRRLARRGGVKRISGLIYEETRGVLKVFLENVIRDAVTYTEHAKRKTVTAMDVVYALKRQGRTLYGFGG
So they observed that in the peptide 1-9 and 10-20 , 66 Da change
Trypsin digestion is done following acetylation, so cleavage is done only after arginine and those lysines. So while comparing wild type H4 with acetylation and the H4 separated from nucleosome after photolysis , there is a difference of 24Da. This treatment generated two modified peptide fragments (amino acids 20−23 and 4−17). The increased mass (24 Da) was consistent with the mass difference between 6 and an acetyl group, suggesting that Lys 20 and at least one of Lys 5, 8, 12, and 16 were involved in the reaction with DOB.
Here they say that there is no peak after the cleavage by Lys C as all of them will be unmodified and will be cleaved at every lysine and arginine and hence from all these they conclude the lys residues are modified at 12, 16, 20 (and in 5,8 they are not able to lack of MS/MS data. In c4AP 8,12,16,20 but not 5 were modified
Digestion of the modified H4 protein by Lys C (which hydrolyzes specifically at the carboxyl side of unmodified lysines) gave rise to a new peak
whose mass was 66 Da greater than that of the unmodified peptide fragment 13−20 (Figure 8). The absence of the corresponding fragment in WT H4 digestion (Figure S24A)was due to cleavage at unmodified Lys 16, indicating that Lys16 was modified by pyrrolone (6) in the NCP.
Lys residues 12,16,20 are modified. H4K16 *– acetylation prevents condensation H4K20*- may block DNA damage and H4K12*- misregulation of dynamics
Sequence of 601 DNA with linker regions extended on both ends. The sites where DOB was generated are shown in red. Sequences in blue represent linker DNA.
Although NCP is a useful model to investigate the behavior of DNA lesions in chromatin, canonical core histone tail – DNA interactions cannot be fully realized. The half-life for DOB89 excision in the NCP and nucleosome were within experimental error of one another. data suggest that the linker DNA did not interrupt the interactions between DOB89 and the histone H4 tail. This observation was corroborated by examining the effect of histone H4 variants on DOB89 reactivity in nucleosomes. The mutagenesis results paralleled those from the corresponding NCP. Substituting five lysine residues in the H4 tail with
arginines (K5,8,12,16,20R) reduced the DOB decomposition rate by ∼7-fold. Moreover, substituting the K5,8,12,16,20R/H18A H4 variant in the nucleosome led to a DOB half-life that was ∼17 times longer than when the WT protein was present. The comparable decreases induced by histone H4 variants with or without linker DNA suggest that histone H3 does not interact with DOB89, even in the absence of nucleophilic residues on the H4 tail.
The C-terminal region of H2A interacts with DNA near the dyad position of a NCP but switches to contact the site near the edge of the core particle in nucleosomes or oligonucleosomes in which linker DNA is present.
The half-life of DOB176 was unaffected by removing either the H3 or H3 and H4 tails, suggesting no interactions between histone tails and the oxidized abasic site at this position. In contrast, the half-life of DOB159 was extended 2-fold in the absence of the H3 tails. Deleting the histone H4 tail had no
additional effect on DOB159 reactivity. These observations suggest that DOB159 within the linker region interacts exclusively with the tail from histone H3. The histone H4 tail was not involved in DOB159 excision, even in the absence of the H3 tail. However, the histone H3 tail accounts for only ∼2-fold of the 135-fold increase in DOB159 reactivity in free DNA.
Acetylases mask negative charge and hence the genes are not made available and the transcriptional rrepression happens during dna damage
The half-life of DOB is the shortest among all the abasic lesions (C4-AP, AP, and L) in 601 NCPs. Its accelerated reactivity in NCPs is more similar to that experienced by C4- AP (up to ∼550-fold) than for either AP (∼100-fold) or L (up to ∼43-fold). Interestingly, a plot of the rate constants
for decomposition of various abasic sites at position 89 in NCPs (kdec) versus reaction in free DNA (kDNA) shows good linearity for AP, C4-AP and DOB, but not L. We suggest that this indicates that AP, C4-AP, and DOB react via a common mechanism but the lactone (L89) does not react via a Schiff-base intermediate
![Introduction
• The initial step of BER involves enzymatic
activities of DNA glycosylases that process the N-
glycosylic bonds linking the target bases and their
deoxyribose sugars
• The central intermediate is an abasic [apurinic ⁄
apyrimidinic (AP)] site in DNA
• In a given day, each cell produces between 10,000
and 50,000 AP lesions
• Oxidized abasic sites eg. C4-AP, L, DOB arise
following hydrogen atom abstraction from the 2'-
deoxyribose ring of a nucleotide](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)