Paper on DNA Repair 036/1443.full?sid=5f09e4ee-ff8b-4f8b- acb2-e79dd986e8f6 .

Paper on DNA Repairhttp://www.sciencemag.org/content/332/6036/1443.full?sid=5f09e4ee-ff8b-4f8b-acb2-e79dd986e8f6

Science 17 June 2011: Vol. 332 no. 6036 pp. 1443-1446

SIRT6 Promotes DNA Repair Under Stress by Activating PARP1

Zhiyong Mao, Christopher Hine, Xiao Tian, Michael Van Meter, Matthew Au,

Amita Vaidya, Andrei Seluanov*, Vera Gorbunova*

UV Absorption of Nucleobases

• Absorption of UV light at 250-270 nm is due to

* electronic transitions

• Excited states of common nucleobases decay

rapidly via radiationless transitions

– Effective photoprotection of genetic material

– No fluorescence from nucleic acids

Some well-characterized nonenzymatic reactions of nucleotides.

Some well-characterized nonenzymatic reactions of nucleotides.

Conformation around N-Glycosidic Bond

• Relatively free rotation can occur around the N-glycosidic bond in free nucleotides

• The torsion angle about the N-glycosidic bond (N-C1') is denoted by the symbol

• The sequence of atoms chosen to define this angle is O4'-C1'-N9-C4 for purine,

and O4'-C1'-N1-C2 for pyrimidine derivatives

• Angle near 0corresponds to syn conformation

• Angle near 180 corresponds to anti conformation

• Anti conformation is found in normal B-DNA

Furanose rings in nucleotides can exist in four different puckered conformations. In all cases, four of the five atoms are in a single plane. The fifth atom (C-2′ or C-3′) is on either the same (endo) or the opposite (exo) side of the plane relative to the C-5′ atom.

Road to the Double Helix• Franklin and Wilkins:

–“Cross” means helix

–“Diamonds” mean

that the phosphate-

sugar backbone

is outside

– Calculated helical

parameters

• Watson and Crick:

– Missing layer means

alternating pattern

(major & minor groove)

– Hydrogen bonding:

A pairs with T

G pairs with C

Double helix fits the data!

Watson, Crick, and Wilkins shared 1962 Nobel Prize

Franklin died in 1958

Complementarity of DNA strands

• Two chains differ in sequence

(sequence is read from 5’ to 3’)

• Two chains are complementary

• Two chains run antiparallel

Figure 5-14 Schematic representation of the strand separation in duplex DNA resulting from its

heat denaturation.

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Thermal DNA Denaturation (Melting)

• DNA exists as double helix at normal temperatures• Two DNA strands dissociate at elevated

temperatures• Two strands re-anneal when temperature is

lowered• The reversible thermal denaturation and annealing

form basis for the polymerase chain reaction• DNA denaturation is commonly monitored by UV

spectrophotometry at 260 nm

Figure 5-15 UV absorbance spectra of native and heat-denatured E. coli DNA.

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Figure 5-16Example of a DNA melting curve.

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Factors Affecting DNA Denaturation

• The midpoint of melting (Tm) depends on base composition– high CG increases Tm

• Tm depends on DNA length– Longer DNA has higher Tm

– Important for short DNA

• Tm depends on pH and ionic strength– High salt increases Tm

Figure 5-17 Variation of the melting temperatures, Tm, of various DNAs with their G + C content.

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Two Near-Complementary DNA Strands Can Hybridize

• Detection of a specific DNA molecule in complex mixture

- radioactive detection

- fluorescent DNA chips

• Amplification of specific DNA

- polymerase chain reaction

- site-directed mutagenesis

• Evolutionary relationships

• Antisense therapy

Diseases often result from failure of DNA repair systems

• colon cancer • cellular ultraviolet sensitivity

• Werner syndrome (premature aging, retarded growth)

• Bloom syndrome (sunlight hypersensitivity)

DNA Repair and Mutations

• Chemical reactions and some physical processes constantly damage genomic DNA– At the molecular level, damage usually involves changes in the structure of one of the strands

– Vast majority are corrected by repair systems using the other strand as a template

– Some base changes escape repair and the incorrect base serves as a template in replication

– The daughter DNA carries a changed sequence in both strands; the DNA has been mutated

• Accumulation of mutations in eukaryotic cells is strongly correlated with cancer; most carcinogens are also mutagens

Molecular Mechanisms of Oxidative and Chemical Mutagenesis

• Oxidative damage• Hydroxylation of guanine• Mitochondrial DNA is most susceptible

• Chemical alkylation• Methylation of guanine

• Cells have mechanisms to correct most of these modifications

Figure 30-51 Types and sites of chemical damage to which DNA is normally

susceptible in vivo. Red, oxidation; blue, hydrolysis; green, methylation.

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73

N

NH

N

N

O

NH2

N

N

H2N

OG C

Normal base pairing

H N

N H

O

ON

N

N

N

O R

N H 2

TO 6 - A l k G

G A

GC

GT

AT

H N

N H

O

ON

N

N

N

O R

N H 2

TO 6 - A l k G

G A

GC

GTGT

ATAT

O6-alkylguanine has a different pattern of H-bond donor and acceptor atoms than the parent guanine base. As a result, it base pairs with T instead of C, giving rise to G A transition after the second round of replication:

Endogenous and exogenous alkylating agents (tobacco smoke, some anticancer drugs). Alkylation destabilizes the glycoside bond and can ultimately lead to backbone breaks.

N N

NN

O

NH2

CH3

O6-methylguanine

AGT-CH2-SH

N NH

NN

O

NH2

AGT-CH2-S CH3

O6-alkylguanine DNA alkyltransferase (AGT)Directly repairs alkylation damage (O6-alkylguanines) by transferring the O6-alkyl group from damaged guanine in DNA to a Cys residue in the AGT active site in a stoichiometric reaction. The protein is inactivated via alkylation and undergoes proteolytic degradation.

AGT protein is highly conserved: helix-turn-helix DNA binding motif the alkylated base is “flipped” out of the helix to enter the hydrophobic alkyl-binding pocket of the protein high metabolic cost for the cell is outweighed by the need to maintain genetic integrity

• UV light induces dimerization of pyrimidines, this may be the main mechanism for skin cancers

• Ionizing radiation (X-rays and -rays) causes ring opening and strand breaking. These are difficult to fix

• Cells can repair some of these modifications, but others cause mutations. Accumulation of mutations is linked to aging and carcinogenesis

Molecular Mechanisms of Radiation-Induced Mutagenesis

Damage of the double helix

• Single strand damage– information is still backed up in the other strand

• Double strand damage– no backup– can cause the chromosome to break up

Molecular Mechanisms of Spontaneous Mutagenesis

• Deamination• Very slow reactions• Large number of residues• The net effect is significant: 100 C ?????

U events /day in a mammalian cell

• Depurination• N-glycosidic bond is hydrolyzed• Significant for purines: 10,000 purines lost/day in a mammalian cell

• Cells have mechanisms to correct most of these modifications.

Figure 30-51 Types and sites of chemical damage to which DNA is normally

susceptible in vivo. Red, oxidation; blue, hydrolysis; green, methylation.

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DNA repair enzymes• a lot of DNA damage → elevated levels of repair enzymes

• extreme change in cell's environment (heat, UV, radiation) activates genes that code DNA repair enzymes– E.g., heat-shock proteins are produced in heat-shock response to high temperatures.

Types of DNA Repair Systems

• Single strand repair• Nucleotide Excision Repair• Base Excision Repair• Mismatch Repair (shortly after

replication)

• Double strand repair• Homologous end-joining• Non-homologous end-joining

Types of DNA Repair Systems

• Mismatches arise from occasional incorporation of incorrect nucleotides

• Abnormal bases arise from spontaneous deamination reactions or via chemical alkylation (alk genes)

• Pyrimidine dimers form when DNA is exposed to UV light

• Backbone lesions occur from exposure to ionizing radiation

Formation of pyrimidine dimers induced by UV light. (b) Formation of a cyclobutane pyrimidine dimer introduces a bend or kink into the DNA

Figure 30-52 The cyclobutylthymine dimer that forms on UV irradiation of two adjacent

thymine residues on a DNA strand.

N H

O

O

H 3C

N

O

O

PO

O

O -

O

N

N H

O

O

C H 3

N H

O

O

H 3C

N

O

O

PO

O

O -

O

N

N H

O

O

C H 3

5'

3'

5'

3'

h

thym ine d im er

Also C -T, C -C dim ers

N H

O

O

H 3C

N

O

O

PO

O

O -

O

N

N H

O

O

C H 3

N H

O

O

H 3C

N

O

O

PO

O

O -

O

N

N H

O

O

C H 3

5'

3'

5'

3'

h

thym ine d im er

Also C -T, C -C dim ers

Photolyase: repairs cyclobutane pyrimidine dimers. Uses the energy of light to catalyze the reversal of the cyclobutane bonds, producing intact DNA. Not very important in mammals.

Nucleotide-excision repair in E. coli and humans

Figure 30-56Base Excision RepairDNA glycosylases hydrolyze the glycosidic bond of their

corresponding altered base to yield an AP site.

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N NH

NN

O

NH2O

O

O

OHOO

O

Abasic site

H2O

Single strand repair

• Nucleotide excision repair– a large multienzyme compound scans the DNA strand for anomalies

– upon detection a nuclease cuts the strand on both sides of the damage

– DNA helicase removes the oligonucleotide

– the gap is repaired by DNA polymerase and DNA ligase enzymes

Base-excision repair pathway

1 DNA glycosylase recognizes damaged base, cleaves N-glycoside bond

2 AP endonuclease cleaves backbone near the AP site.

3 DNA pol I initiates repair synthesis from the free 3′ OH at the nick, removing and replacing the damaged strand.

4 Nick sealed by DNA ligase.

Extrahelical Damaged Base Recognition by DNA Glycosylase Enzymes

Chemistry – A European JournalVolume 14, Issue 3, pages 786-793, 15 NOV 2007 DOI: 10.1002/chem.200701501http://onlinelibrary.wiley.com/doi/10.1002/chem.200701501/full#fig5

Intermediates on the base flipping pathways of hOGG1 and UNG.a) The exo-site complex of hOGG1 with an extrahelical guanine (blue) obtained by disulfide crosslinking technology (left). The fully extrahelical complex with 8-oxoG is shown on the right for comparison. b) The early exo-site complex of hUNG with an extrahelical thymine (blue) obtained using the reaction coordinate tuning method. The fully extrahelical complex with uracil is shown on the right.

Methylation and mismatch repairReally only understood well in E.coli.

The methylation occurs at the N6 of adenines in (5′)GATC sequences.(palindrome)

Dam=DNA adenine methylation

Double strand repair

• Homologous end-joining– damaged site is copied from the other chromosome by special recombination proteins

Double strand repair

• Nonhomologous end-joining– only in emergency situations

– two broken ends of DNA are joined together

– a couple of nucleotides are cut from both of the strands

– ligase joins the strands together

Cell Cycle and DNA repair

• Cell cycle is delayed if there is a lot of DNA damage.

• Repairing DNA as well as signals sent by damaged DNA delays progression of cell cycle.

This ensures that DNA damages are repaired before the cell divides

It is interesting to note that NAD+ is required as substrate for generating ADP-ribose monomers. The overactivation of PARP may deplete the stores of cellular NAD+ and induce a progressive ATP depletion, since glucose oxidation is inhibited, and necrotic cell death. In this regard, PARP is inactivated by caspase-3 cleavage (in a specific domain of the enzyme) during programmed cell death.

PARP enzymes are essential in a number of cellular functions, including expression of inflammatory genes: PARP1 is required for the induction of ICAM-1 gene expression by smooth muscle cells, in response to TNF. Activity PAR is synthesized using nicotinamide (NAM) as the leaving group. This leaves a pyrophosphate as the linking group between ribose sugars rather than single phosphate groups. This creates some special bulk to a PAR bridge, which may have an additional role in cell signaling.

Role in repairing DNA nicks

One important function of PARP is assisting in the repair of single-strand DNA nicks. It binds sites with single-strand breaks through its N-terminal zinc fingers and will recruit XRCC1, DNA ligase III, DNA polymerase beta, and a kinase to the nick. This is called base excision repair (BER). PARP-1 is also known for its role in transcription through remodeling of chromatin by PARylating histones and relaxing chromatin structure, thus allowing transcription complex to access genes.

Sirtuin or Sir2 proteins are a class of proteins that possess either histone deacetylase or mono-ribosyltransferase activity. Sirtuin activity is inhibited

by nicotinamide, which binds to a specific receptor site

Crystallographic structure of yeast sir2 (rainbow colored cartoon, N-terminus = blue, C-terminus = red) complexed with ADP (carbon = white, oxygen = red, nitrogen = blue, phosphorus = orange).

Supplementary Figure 1. Constructs integrated in the reporter cell lines for detectingNHEJ and HR. A, NHEJ reporter cassette. The construct consists of a GFP genecontaining an intron from the rat Pem1 gene, interrupted by an adenoviral exon (Ad).The adenoviral exon is flanked by I-SceI recognition sites in inverted orientation forinduction of DSBs. In the starting construct the GFP gene is inactive. Induction of aDSB by I-SceI followed by NHEJ reconstitutes the functional GFP gene. SD, splice donor; SA, splice acceptor; shaded squares, polyadenylation sites. B, HR reportercassette. The construct consists of two mutated copies of GFP-Pem1. In the first copy of GFP-Pem1 the first GFP exon carries a deletion of 22 nt and an insertion of two I-SceI recognition sites in inverted orientation. The 22 nt deletion ensures that GFP cannot be reconstituted by an NHEJ event. The second copy of GFP-Pem1 is lacking the ATG andthe second exon of GFP. Upon induction of DSBs by I-SceI, gene conversion eventsreconstitute the GFP gene. C, Incompatible DNA ends generated by digestion of twoinverted I-SceI sites.

Fig. 1 SIRT6 stimulates DSB repair.

Z Mao et al. Science 2011;332:1443-1446Published by AAAS

SIRT6 stimulates DSB repair. (A) Overexpression of SIRT1, -2, -6, and -7

in human fibroblasts. Immunoblotting with sirtuin-specific antibodies after transfection with a sirtuin-expressing vector or a control vector encoding hypoxanthine-guanine phosphoribosyltransferase (pControl).

(B) (B) Effect of sirtuin overexpression on the efficiency of NHEJ and HR, measured as described in fig. S1. The efficiency of DSB repair was scored in untreated cells (open bars), cells pretreated with 1 mM paraquat for 16 hours (black bars), or cells treated with paraquat and 5 mM nicotinamide for 16 hours (red bars). GFP, green fluorescent protein.

(C) (C) SIRT6 overexpression accelerates the disappearance of γH2AX foci after treatment with 1 mM paraquat for 16 hours. Data represents an average of at least 50 nuclei.

(D) (D) Immunoblot showing induction of endogenous SIRT6 protein levels by oxidative stress. Human fibroblasts were treated with paraquat for 16 hours.

Fig. 2 Oxidative stress results in early recruitment of SIRT6 to DNA breaks.

Z Mao et al. Science 2011;332:1443-1446

Published by AAAS

Oxidative stress results in early recruitment of SIRT6 to DNA breaks. ChIP analysis showing kinetics of SIRT6 recruitment to Alu sequences after 8 Gy of γ-irradiation (IR) (A) and sequences flanking I-SceI–induced DSB after transfection with I-SceI expression vector

(B). Asterisks indicate values significantly different from corresponding zero time points (P < 0.05). Error bars indicate SD; n = 5. Control ChIP with SIRT6−/− cells is shown in fig. S7. IgG, immunoglobulin G.

Fig. 3 Deacetylation and mono-ADP-ribosylation activities of SIRT6 are required to stimulate DSB repair.

Z Mao et al. Science 2011;332:1443-1446

(A) Immunoblot showing that S56Y and R65A mutations abolish the H3K9 deacetylation activity of Sirt6 and appear to exert a dominant-negative effect.

(B) In vitro assay showing that S56Y and G60A mutations abolish mono-ADP-ribosylation activity of SIRT6. NAD+, nicotinamide adenine dinucleotide.

(C) SIRT6 mutants for deacetylation and/or ribosylation activities have reduced ability to stimulate NHEJ and HR. Untreated cells (open bars) or cells treated with paraquat (black bars) were transfected with SIRT6-expressing vectors or pControl.

Fig. 3 Deacetylation and mono-ADP-ribosylation activities of SIRT6 are required to stimulate DSB repair.

Z Mao et al. Science 2011;332:1443-1446

(A) Immunoblot showing that S56Y and R65A mutations abolish the H3K9 deacetylation activity of Sirt6 and appear to exert a dominant-negative effect.

(B) In vitro assay showing that S56Y and G60A mutations abolish mono-ADP-ribosylation activity of SIRT6. NAD+, nicotinamide adenine dinucleotide.

(C) SIRT6 mutants for deacetylation and/or ribosylation activities have reduced ability to stimulate NHEJ and HR. Untreated cells (open bars) or cells treated with paraquat (black bars) were transfected with SIRT6-expressing vectors or pControl.

Fig. 4 SIRT6 interacts with PARP1 and stimulates its poly-ADP-ribosylation activity.

Z Mao et al. Science 2011;332:1443-1446

(A) Analysis of mono-ADP-ribosylated proteins in the WT and SIRT6−/− MEFs stressed with paraquat for 16 hours. Cells were transfected with biotinylated NAD, and poly-ADP-ribosylated proteins were cleared away with PAR antibodies.

(B) Immunoblotting of the extracts in (A) with PARP1 antibodies indicated that the 120-kD band is Parp1.

(C) SIRT6 interacts with PARP1. Human fibroblasts were treated with 1 mM paraquat. Cell lysates were immunoprecipitated with SIRT6 antibodies in the presence of ethidium bromide (EtBr) followed by Western blotting with PARP1 antibodies.

(D) PARP1 K521 is essential for activation of NHEJ by SIRT6. An NHEJ assay was performed in PARP1−/− MEFs containing an integrated NHEJ reporter. Cells were transfected with SIRT6 and/or PARP1 or PARP1 mutants. Both SIRT6 and PARP1 are required for the stimulation of repair. PARP1 Y889C is a catalytically inactive PARP1. PARP1 DEEKKK contains mutations in all six poly-ADP-ribosylation sites. PARP1 DEEKK contains the same mutations, except at K521. Asterisks indicate values significantly different from control (P < 0.01). (E) PARP1 lacking the catalytic domain is mono-ADP-ribosylated by SIRT6 in vitro, whereas K521A is not.

(F) In vitro assay of PARP1 poly-ADP-ribosylation activity showing that PARP1 is stimulated only by SIRT6 mono-ADP-ribosylation activity. (G) Stimulation of NHEJ and HR by SIRT6 is abolished by PARP1 inhibitors 3-ABA or PJ34.

(D) PARP1 K521 is essential for activation of NHEJ by SIRT6. An NHEJ assay was performed in PARP1−/− MEFs containing an integrated NHEJ reporter. Cells were transfected with SIRT6 and/or PARP1 or PARP1 mutants. Both SIRT6 and PARP1 are required for the stimulation of repair. PARP1 Y889C is a catalytically inactive PARP1. PARP1 DEEKKK contains mutations in all six poly-ADP-ribosylation sites. PARP1 DEEKK contains the same mutations, except at K521. Asterisks indicate values significantly different from control (P < 0.01). (E) PARP1 lacking the catalytic domain is mono-ADP-ribosylated by SIRT6 in vitro, whereas K521A is not.

(F) In vitro assay of PARP1 poly-ADP-ribosylation activity showing that PARP1 is stimulated only by SIRT6 mono-ADP-ribosylation activity.

(G) Stimulation of NHEJ and HR by SIRT6 is abolished by PARP1 inhibitors 3-ABA or PJ34.

• Abstract• The efficient enzymatic detection of damaged

bases concealed in the DNA double helix is an essential step during DNA repair in all cells. Emergent structural and mechanistic approaches have provided glimpses into this enigmatic molecular recognition event in several systems. A ubiquitous feature of these essential reactions is the binding of the damaged base in an extrahelical binding mode. The reaction pathway by which this remarkable extrahelical state is achieved is of great interest and even more debate.

X-Ray structure of human uracil–DNA glycosylase (UDG) in complex with a 10-bp DNA containing a U·G base pair. Mismatch flipped out. DNA helix stabilized by aa plug.

Catalysis is by GAB, but activity is not abolished by loss of the acid residue in the active site. Conformational strain helps drive the reaction!!aa plug prevents base from flipping back into helix.

EH Rubinson et al. Nature 000, 1-6 (2010) doi:10.1038/nature09428

Base excision repair of alkylated DNA by AlkD in Bacillus cereus.

a, AlkD catalyses the hydrolysis of the N-glycosidic bond to liberate an abasic site and free nucleobase (100X rate increase over spontaneous depurination). The enzyme is specific for positively charged N3-methyladenine (a) and N7-methylguanine (b). c, d, Structures used to trap AlkD in complex with alkylated and abasic DNA. e, Crystal structure of AlkD bound to 3d3mA-DNA. Each of the six HEAT repeats is coloured red-to-violet. The DNA is coloured silver with the 3d3mA nucleotide colored magenta.

Lack of + adds stability THF

Resembles apurinc DNA

alkD consists of HEAT repeats

• HEAT: domain found in a number of cytoplasmic proteins including Huntingtin, elongation factor 3 (EF3), protein phosphatase 2A (PP2A), and the yeast PI3-kinase TOR1and form rod-like helical structures

• A substantial number of HEAT repeats have been detected in proteins involved in translation including all eIF4Gs!

Figure S3. DNA binding by the HEAT repeats of AlkD. Electrostatic surface potential (blue, positive; red, negative) of AlkD showing a high degree of positive charge within the concave DNA binding cleft. Structure based sequence alignment of HEAT repeats. Residues that contact the DNA in both substrate (3d3mA•T, G•T) and product (THF•T, THF•C) complexes are highlighted yellow, residues contacting the DNA in only substrate or only product are highlighted blue and magenta, respectively.

EH Rubinson et al. Nature 000, 1-6 (2010) doi:10.1038/nature09428

Crystal structures of AlkD in complex with 3d3mA-DNA (a) and THF-DNA (b).

The modified 3d3mA and tetrahydrofuran (THF) nucleotides are colored blue, and the opposing thymine is magenta.

DNA positioned along +charged concave surface of alkDContacts 10 bp

Contacts cluster around mismatch

Fewer contacts on lesion strand

Both 3m3A and THF reside on the face of the DNA duplex NOT in contact with the protein whereas the compelement is nestled in the enzyme cleft.

This THF trapped complex shows the abasic site rotated 90o around the PD backbone where it is totally solvent exposed!!

Opposing T has slipped out of helix into DNA minor groove!!

DNA duplex has collapsed to retain stacking interactions.

Backbone distorted

NO aa PLUG

Recognition of DNA damage by AlkD. a, 3d3mA-DNA (substrate) complex.

b, THF-DNA (product) complex.

Dashed arrows denote displacement of THF and opposing thymine from their positions in B-DNA. Hydrogen bonds are shown as dashed lines. Views are down the DNA helix axis.

Remodelling of a G•T wobble base pair by AlkD.

a, AlkD–G•T-DNA complex viewed down the helical axis. b, The structure of a G•T wobble base pair in DNA alone is superimposed onto the AlkD–G•T complex. Steric clashes between the protein and DNA are highlighted by yellow stars, and disrupted hydrogen bonds (dashed lines) are shown by a red X. c, Relative single-turnover rates (kst) of 7mG excision from a 25mer oligonucleotide duplex by wild-type AlkD and the indicated AlkD mutants.

AlkD seems to detect DNA duplex destabilization rather than specifically recognizing modified bases. Protein restructures

the wobble and disrupts base stacking.

Solvent exposure increases the lifetime that spontaneous

depurination is likely to occur!!!

The phosphate groups on the DNA may participate in the rate

enhancement by positioning water molecules for solvent attack on the

glycoside bond.

Figure S12. Proposed mechanism for how AlkD facilitates hydrolysis of N3 and N7-alkylpurines by distorting the DNA backbone.

AlkD captures the DNA in an orientation that holds the orphaned base next to the protein and exposes the lesion to a hydrolytic environment. The distorted DNA conformation is stabilized not by a side chain plug, but by stacking of flanking base pairs as a result of both lesion and orphaned base flipping. Phosphate (gold “P”) assisted hydrolysis could occur either by positioning of the water molecules adjacent to the C1' carbon in a dissociative hydrolysis reaction, or through stabilization of an oxocarbenium ion intermediate

Chapter 8: Summary

• Function of nucleotides and nucleic acids• Names and structures of common nucleotides• Structural basis of DNA function • Reversible denaturation of nucleic acids• Chemical basis of mutagenesis

In this chapter, we learned about: