UNIT III: DNA Replication, Recombination and Repair (Sandip Das)
Module 16
3.3 DNA damage and repair
3.3.1 Nucleotide Excision repair system pathway Eukaryotic pathway (NER pathway)
Prokaryotic pathway or Excision repair in bacteria 3.3.2 Base-excision repair pathway (BER pathway)
3.3.3 UV damage and Photo-reactivation or light repair pathway 3.3.4 Error-prone repair / Translesion synthesis pathway
3.3.5 Chromatin structure and DNA damage
Glossary
Useful Web resources Further Readings:
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Introduction:
DNA as genetic material is subjected to constant wear and tear due to various “faults” in the enzymatic process that occurs during replication process; apart from replication induced damage, DNA is also subjected to various stresses such as oxidative damage (because of reactive oxygen and nitrogen species), lipid peroxidation and environmental damage because of UV radiation. Repair of DNA is imperative as continued accumulation of damage in the DNA may result in disorders (please note that damage only in the germ line DNA will be inherited).
Various pathways exists that are employed by the cell to repair damaged DNA; for example mismatch repairs are carried out by the nucleotide excision repair and base excision repair pathway;and the recombination –repair system that is involved in double stranded break (DSB). The recombination repair system is discussed in the module on Recombination.
The following sections will provide an overview of the various DNA damage repair pathways known.
3.3.1 Nucleotide repair system pathway / Nucleotide excision repair or NER pathway Eukaryotic pathway
Removal or Excision of a sequence that includes the damaged nucleotides (termed as Nucleotide excision repair-NER) is the major pathway for removal and repair of lesions in DNA formed by UV and mutagens. NER pathway is involved whenever any helical distortion or modification in DNA chemistry such as intra-strand cross, thymine dimers are to be repaired. In other words, NER pathway targets lesions that lead to helix distortion and impairs transcription and replication.Two major pathways are responsible for NER. These two are termed as Global Genome NER or GG-NER(that carries out repairs in the transcriptionally silent regions of the genome) and Transcription Coupled NER or TC- NER(that repairs lesions in actively transcribed strand of DNA).
The two pathways of NER differ primarily with respect to the damage recognition mechanism. Once initiated, both GG-NER (Figure 1) and TC-NER (Figure 2) require a core set of common factors to carry out the repair process which is nearly identical.For example, in GG-NER, the damage is recognized by a protein termed as XenodermaPigmentosa Complementation Group C (XPC) that binds to the non-damaged strand opposite to the lesion; whereas in TC-NER, damage is recognized when the movement of RNA polymerase is stalled or blocked due to altered DNA structure (helical distortion, addition of bulky
residues, thymine dimers etc).Repair in the GG-NER pathway is initiated by the UV- damaged DNA binding protein or UV-DDB (RAD23A).Repair in the TC-NER pathway is initiated by RNA-polymerase that is stalled at the lesion in the DNA with the help of TC- NER pathway specific proteins such as Cockayne Syndrome A (CSA; also annotated as ERCC8-excision repair cross-complementation group 8), Cockayne Syndrome B(CSB;
ERCC6-excision repair cross-complementation group 6) and XAB2 (XPA binding protein 2).
XPC binds as XPC-RAD23B complex(Figure 3) in the GG-NER repair pathway which then interacts with the multi-subunit transcription factor- TFIIH. The XPB and XPD subunits of TFIIH, which are helicases, then unwind the DNA. The XPD protein “slides”along the DNA until it stalls at the site of lesion.Subsequently, other proteins-namelyXPA and XPG with another protein RPA (Replication protein A) aid in further marking the site of DNA damage.
XPG protein is an endonuclease which nicks the DNA 5’ to the lesion, but only after the ERCC1-XPF complex has been recruited after interaction with XPA. Once a nick has been made, DNA pol δ (delta), pol κ (kappa) and pol ε (epsilon) initiate repair synthesis, and eventually the damaged region is released by nicking towards the 3’of the lesion by XPG.
The nick is finally sealed by DNA ligase thus completing the repair process.
In summary, NER occurs in six discrete yet overlapping steps (Figure 4)–
Lesion recognition by XPC and RNA polymerase
Unwinding of duplex DNA using helicase property of XPB
Incision / nick towards 5’of lesion by XPG
Repair synthesis using DNA pol δ (delta), pol κ (kappa) and pol ε (epsilon)
Incision / nick towards 3’of lesion and removal of damaged DNA
Sealing of nick using Ligase
NER is thought to be one of the most versatile DNA damage repair pathways as one set of enzymes can recognize a wide range of substrates generated as a result of various mutational events-chemical, environmental and UV induced. A few examples of such substrates are UV induced cyclobutane pyrimidine dimers (CPD) and pyrimidine 6-4 pyrimidone photoproducts (6-4 PP), benzo(a)pyrene formed as a result of environmental mutagens, cyclopurines (endogenous oxidative lesions),diol-epoxide, aromatic amines such as acetyl-amino-fluorene, aflatoxin, nitrosoamines such as MNNG (Methylnitronitrosoguanidine), and 4-nitro-quinoline oxide.Apart from these substrates, NER can also repair mutations caused by cross-linking
chemicals such as Cisplatin (a cancer chemotherapeutic drug) those that cause intra-strand and inter-strand crosslinks.All these modifications cause bulky lesions that destabilize the DNA duplex.
A rare autosomaldisorderXerodermapigmentosa (XP)and Cockayne Syndrome are outcomes of the non-functional NER pathway.
Figure 1
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Figure 4:
Excision repair in bacteria
The nucleotide excision repair pathway in bacteria/prokaryotes is regulated via action of UVrABC endonuclease complex of enzymes. This complex consists of UVrA, UVrB, UVrC and UVrD (which is a DNA helicase). AUVrA dimer complexes with UVrB to form aUVrA- UVrBtrimer complex. The UVrA protein in the UVrA-UVrB complex is instrumental in locating the damaged nucleotides such as bulky bases or UV-induced dimers. The UVrB than binds to the damaged site and the UVrA protein dissociates in an ATP dependent manner and UVrC protein binds to form a UVrBC complex with the DNA. This UVrBCcomplex then
cleaves the DNA at two positions, between seven-eight nucleotides upstream of the 5’end and five-four nucleotides downstream of the 3’end thus creating a 12 nucleotide gap. The endonucleotic cleavage is catalyzed by UVrB (towards the 3’end i.e. downstream end) and UVrC (towards the 5’ end i.e. upstream end). The UVrC then dissociates and subsequently, the fourth partner, UVrD or DNA helicase breaks the H-bond between the complementary nucleotides, unwinds the DNA and releases the 12-nt DNA. DNA polymerase I is responsible for synthesis of the missing bases and in the process dislodges UVrB. Finally, DNA ligase seals the nick and restores the original DNA sequence (Figure 5).
Figure 5:
3.3.2 Base-excision repair pathway
In contrast to the NER pathway that repair bulky lesions, base excision repair (BER) pathway is involved in repair of smaller lesions that do not cause helical distortions.In addition, whereas NER excises out the entire stretch of sequences harbouringaltered/damaged
nucleotide, instead BER pathway is critical in restoring original nucleotide by removing damaged bases so that mis-pairing during replicationleading to mutations can be avoided.
Examples of such damaged or mis-incorporated bases include:
a. Oxidized bases:8-oxoguanine; 2,6-diamino-4-hydroxy-formamidopyrimidine b. Alkyated bases: 3-methyl adenine; 7-methyl guanine
c. Deaminated bases: Hypoxanthine; Xanthine d. Uracil
Figure 6:
Coordinated action is required between two enzymes DNA Glycosylase that recognizes and the damaged base and creates apurinic (AP) sites; Figure 6) by cleaving the N- glycosidic bond to remove the damaged base; AP endonucleasethen cleaves the AP site to create several types of free ends. Two types of DNA glycosylases are known-
monofunctional type and bifunctional type. The monofunctional type leaves the intact AP site in DNA as it cleaves the glycosidic bond releasing the damaged base; bifunctional type on the other hand, cleaves both glycosidic bond and phosphodiester bond via AP-lyase activity and leaves incised DNA and the free base. UNG (Uracil N-Glycosylase; human) and UDG (Uracil DNA glycosylase; E.coli; Figure 7) are examples of monofunctionalglycosylases.
Other examples of monofunctional DNA glycosylasesareTDG(Thymine DNA glycosylase) and MBD4 (Methyl CpG binding protein), both from human which areinvolved in repairing T:Gmis-pairing.DNA glycosylases employ a “base flipping” mechanism to access the damaged base.
An AP site is a position in the DNA that has neither purine or a pyrimidine residue and is also known as abasic site. AP endonucleases(Figure 8) are involved in removal of AP sites by strand incision that leaves 3-Phosphate and a 5’-Phosphate end; or a3’-Hydroxyl and 5’- deoxyribose Phosphate end; or a 3-phosphoglycoaldehyde and 5-Phosphate end. AP endonuclease is also involved in modification/repair of 3’ends such that free 3’-OH is generated that will allow DNA polymerase to catalyse addition of new bases.
Figure 7:
Figure 8:
Once a damaged base has been recognized by DNA glycosylase and cleaved by AP endonuclease, either a short-patch or a long patch BER mechanism repairs the gap. Short patch repair involves addition of single base, whereas long-patch repair involves addition of
2-10 nucleotides. Short patch repair is catalysed by DNA polymerase β (beta) with DNA polymerase λ (lambda) playing a compensatory role.AP endonuclease and DNA polymerase β (beta) form a ternary complex that is correlates with higher levels of 5’-deoxyribose phosphate lyase activity of AP endonuclease and gap filling activity of DNA pol β (beta) (Liu et al. 2007). In contrast long-patch repair involves displacement synthesis catalysed by DNA polymerase δ (delta) and DNA polymerase ε(epsilon) along with PCNA (Proliferating Cell Nuclear Anitgen), a processivity factor involved in DNA replication. Displacement synthesis leads to formation of a single sranded “flap” which is removed by an enzyme termed as Flap Endonuclease. Once the flap is removed, the nick is joined by DNA ligase I to complete the repair process; in short-patch repair, DNA ligase III seals the nick(Figure 9).
Figure 9:
3.3.3 UV damage and Photo-reactivation or light repair pathway
Ultra-Violet rays are a powerful mutagen and acts by breaking double bond of pyrimidine bases. Once the double bond is broken, free base (still attached to the sugar) is free to react with adjacent nucleotide. Two types of dimers may be recognized-four-membered ring structures termed as Cyclobutane pyrimidine dimers (CPD; Figure 10), and (6-4) Pyrimidone Photo products or (6-4) PP. CPD lesions account for nearly 70%-80% of all UV damages with the rest being (6-4) PP. In case the adjacent / next base is also a pyrimidine (thymine), the two bases form covalent bond and form a dimer (thymine dimer; Figure 11,Figure 12). The photoreactivation process involving the enzyme Photolyase repairs the damage (Figure 11).Photolyase catalyzed DNA repair is a light dependent reaction and is known as photo-reactivation and requires light in the 320-400nm (UV-A), and 400-500 nm (blue) range. Various types of photolyase are responsible for recognizing different substrates- CPD photolyasesor DNA photolyases recognize and repair CPD lesions; (6-4)Photolyases repairs (6-4) PP-damages.These lesions block both DNA and RNA polmerase and thus threaten the fidelity of replication and transcription. All photolyases contain non-covalently bound Flavin Adenine Dinucleotide (FAD) as cofactor; Apart from FAD, Photolyases contain either a folate or a deaza-flavin[5,10-methenyltetrahydrofolylpolyglutamate (MTHF), or 8- hydroxy-5-deazaflavin (8-HDF)] as a second chromophore. The presence of these second chromophore increases the wavelength absorbance range for photolyase activity. Falvin containing photolyases act in 377-410 nm range, whereas deaza-flavin containing photolyases act maximally in 430-460 nm range. There are evidences to suggest that a third type of photolyase which do not contain a second chromophore may also exist such as those from Thermusthermophilus.
The enzyme photolyaseis activated by absorbing light and binds to the damaged CPD regions including 6-8 bases surrounding the region (Figure 11). The CPD or damaged bases are
“accommodated” inside the enzyme active site pocket where FAD (the redox-active co- factor) and a second chromophoreis also bound. Light driven reduction of FAD into FADH- provides electron to break the H-bond of the CPD (Thymine dimer; pyrimidine dimers). Once the H-bonds between the neighbouring bases are broken, the pyrimidine bases can form H- bond with their corresponding complementary bases on the opposite strand thus restoring the damage.
Figure 10:
Figure 11:
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Figure 13:
3.3.4 Error-prone repair / Translesion synthesis pathway / SOS pathway
Error prone repair pathway or Translesion synthesis pathway occurs when the DNA polymerase stalls during replication because of un-repaired or damaged DNA such as thymine dimers or apurinic sites. This causes specialized DNA polymerase broadly termed as
“Translesion DNA polymerases”(members of Pol γ family) to undertake the repair function.
Examples of such translesion DNA polymerases are DNA Pol II, Pol IV and Pol V (all three from E.coli), and nearly 10 DNA polymerases from eukaryotes (humans- REV1, REV3, Pol η [eta], Pol ι [iota], pol κ [kappa]; S.cerevisae/yeast- Rev1p, Rev3p; and several others). Such translesion DNA polymerases have low fidelity, often incorporate incorrect bases and thus are also referred to as Error-prone repair. It appears that the fidelity of these polymerases is
related to the nature of damaged base/s. For example, DNA pol eta (η; encoded by RAD30) performs high-fidelity repair at sites affected by UV irradiation induced thymine dimers (CPD) whereas the same site is acted upon by DNA pol iota (ι) or Pol V in an error-prone manner.REV1 from yeast prefers exclusive incorporation of cytosine opposite Abasic site.
Translesion DNA polymerase have the ability to copy damaged DNA and perform by-pass DNA replication; these are characterized by high error rates, often as high as 10-1 to 10-3; tendency of mis-pairing and the ability to perform polymerization using mis-primed template, aberrant primer ends (instead of 3’-OH), and damaged DNA.
In E.coli, two molecules of UmuD’ combines with one molecule of UmuC to form translesion DNA pol V; this heterotrimeric complex is the error-prone DNA pol V. Once a DNA Pol III containing replisome complex (including sliding clamp, γ-clamp loading complex) encounter a lesion, the tranelseion DNA polymerases (heterotrimeric DNA pol V mutalong with RecA-ATP, SSB assemble at the damaged site via interaction with sliding clamp (β-subunit) and performs insertion and extension of base (translesion synthesis). After performing the translesion synthesis, Pol V is replaced again by the DNA pol III which continues the high fidelity replication.Apart from Pol V, E.coli can also recruit Pol II and Pol IV for TLS.
In eukaryotic system, translesion synthesis (TLS)is performed by several DNA pols (atleast five), sometimes acting individually or sometimes via sequential action of two DNA polymerases.Some of these are DNA pol η(Figure 14), pol ι, pol κ, pol ζ4 and REV1; in addition, other DNA pols such as β, Pol λ, Pol θ and Pol ν may also take part in TLS. DNA pol ζ (zeta; encoded by REV3 and REV7) performs TLS on UV induced damaged DNA; Pol η can perform TLS on damaged bases caused by oxidative reaction such as 8-oxoG (7,8- dihydro oxoguanine) and incorporates Adenine thus causing G:C->A:T mutation. DNA pol η can perform both insertion of nucleotide opposite damaged base and extension such as in CPD (caused as a result of UV irradiation); however, in certain cases, the two steps are performed by two different translesion synthesis DNA polymerases. For example, in cases of bypassing TT dimer, DNA pol η can insert a base opposite the 3’-T and then DNA pol ζ extends from the base; similarly, in (6-4) PP, DNA pol η inserts a G opposite the 3’T and the DNA pol ζ extends from the base. The action of Pol η in the sequential bypass-synthesis can also be replaced by Pol ι in repair of (6-4) PP.Apart from CPD lesions, sequential action is also observed in repair of lesions caused by abasic sites. For example, DNA pol η and REV1 inserting a G or C, respectively whereas DNA pol δ inserting an A. These bases are then extended by DNA pol ζ. Interaction with PCNA is essential for targeting DNA pol η to the
stalled replication machinery and stimulating the insertion of a base opposite an abasic site (Haracska et al. 2001).
Structural analysis of DNA pol η revealed that the active site can accommodate two template nucleotides instead of one in the classical DNA pols (Trincao et al. 2001). In addition, it contains a PAD (Polymerase associated domain) domain which is absent other classical DNA polymerases (Palm, Thumb and Finger domains only). The PAD joins the thumb by a flexible stretch which increases the binding surface of the Pol η and may help in stabilizing the Pol η and DNA complex(figure 14).
Figure 14:
3.3.6 Chromatin structure and DNA damage
DNA damage repair is dependent on changes in chromatic structure that provides access to the repair machinery. Such changes in chromatin structure are driven by two mechanisms: i) histone modification, and ii) histone / nucleosome displacement(Dinant et al. 2008).
The major histone modifications that have been implicated in DNA damage repair include methylation, phosphorylation, acetylation and ubiquitination. For example, methylation of lysine residues in histones are part of DNA damage response. Generally, H3K4-me, H3K36- me and H3K79-me are linked to transcriptional activation whereas H3K9me, H3K27-me and H4K20-me are marks of transcriptional repression.Methylation marks linked to UV irradiation include those on H3K79 and H4K20.Phosphorylation of serine-139 (humans; S- 129 in yeast) on H2AX is a result of DNA double strand breaks (DSB) and this can occur over a large distance of upto 2x106bp, thus creating a chromatin mark that functions as Irradiation Induced Foci (IRIF). The IRIF is then used by DNA damage response factor proteins/enzyme and co-factors to assemble and is an important activator of cell-cycle checkpoint. Other than S-139/S-129, S-122 on H2AX is de-phosphorylated upon UV- irradiation whereas other damages such as NER and non-homologous end joining (NHEJ).
H3S10 and H3T11 undergo de-phosphorylation upon UV irradiation and phosphorylation after the damage has been repaired. As reduced phosphorylation of H3S10 and H3T11 are coupled with transcription repression, de-phosphorylation of H3S10 and H3T11 can lead to suppression of transcription in region with damaged bases. The third form of histone modification is acetylation and it is known that hyperacetylationof histone occurs as a result of UV irradiation. Acetylation of lysine residues on all four histone proteins are linked to DNA damage response and the acetylation are catalyzed by two histone acetyl transferases (HATs)-Gcn5 and p300. Addition of ubiquitin moiety to lysine residues on H2A and H2B is related to gene activation and repression (H2A-ub causes repression; H2B-ub causes activation and repression). Conjugation of Ubi to lysine at position 123 on H2B (in yeast) is linked to UV damage, and activation of checkpoint kinase in affected in case the Ubiquitination (H2BK123) fails to occur. Similar ubiquitination on H2A has been reported in mammalian cells in response to UV damage. In addition, in mammalian cells, ubiquitinantion of H3 and H4 histones that destabilize nucleosome occurs. This may facilitate and allow assembly of NER complex on damaged DNA.Ubiquitination of H2A and H2AX also occurs as a result of double stranded breaks (DSB).
ATP-dependent chromatin remodeling leading to displacement of histones is also involved in DNA damage repair. For example SWI/SNF has been shown to be involved in NER, in-vitro (Hara andSancar 2002). CSB protein (Cockayne Syndrome B) can cause displacement of regularly spaced nucleosome particles in a TC-NER reaction in vitro; and CSB protein has been shown to have chromatin remodeling activity. Rad-16 which has Snf2 like domain (a domain found in chromatin remodelers) is involved in GG-NER.
Table: List of Histone modification and chromatin remodelers associated with DNA damage repair
Histone Modification DNA repair
H2AXS139P NER
H2A-ub NER
H3K9ac/H3K14ac NER
H3K79me NER
H4K20me NER
H3/H4-ub NER
Chromatin remodeler
ACF NER
SWI/SNF NER
CSB NER
CAF1 NER
Figure 15:
