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14
New Players in Recognition
of Intact and Cleaved AP Sites:
Implication in DNA Repair in Mammalian Cells
Svetlana Khodyreva and Olga Lavrik
Institute of Chemical Biology and Fundamental Medicine,
Siberian Branch of the Russian Academy of Sciences,
Russia
1. Introduction
The apurinic/apyrimidinic (AP) sites (called also abasic sites) are common lesion in genomic
DNA, arising at a frequency of 10,000 to 50,000
lesions per mammalian cell per day (Lindahl,
1993). Unrepaired AP sites present mutagenic and cytotoxic consequences to the cell (Wilson;
& Thompson, 1997). Most of the abasic sites are believed to result directly from spontaneous
depurination, or indirectly from deamination of cytosine to uracil, which is then eliminated by
uracil glycosylases. AP sites also result from hydrolysis of oxidized or alkylated bases by
lesion-specific glycosylases during the early stage of base excision repair (BER) (McCullough et
al., 1999). AP sites in isolated DNA are rather stable, but can be converted to single-strand
breaks by alkali treatment, heating or nucleophilic attack at the aldehydic C1’ group (Burrows
& Muller, 1998). Intact abasic sites are noncoding lesions and in vivo can be stable enough to be
mutagenic during DNA replication (Loeb & Preston, 1986). To protect genome integrity,
eukaryotic organisms have robust enzyme activities, mainly APE1 in mammalian cells (Wilson
& Barsky, 2001), that recognize abasic sites and cut the DNA backbone initiating the repair
process. The continuous generation and repair of AP sites results in a steady-state levels of AP
sites in mammalian cells in the range of approximately 1 site per 10
6
nucleotides (Atamna et
al., 2000, Mohsin Ali et al., 2004). The number of AP sites can increase dramatically under
stressful conditions such as X-ray or UV light irradiation or oxidative and alkylating agent
exposure (Atamna et al., 2000). Considering the ubiquity of these lesions, it is reasonable to
assume that wide range of cellular proteins can interact with abasic sites depending on the
physiological state and stages of cell cycle.
The chapter is devoted to search of previously unrecognized proteins capable to interact
with intact or cleaved AP sites. We mainly focused on proteins that form Schiff base upon
this interaction. In most cases, these proteins are able to process AP sites although less
efficiently than previously known counterparts. The biological role of these interactions in
providing of backup pathways of DNA repair processes is also discussed.
2. Proteins that recognize AP sites
In genomic DNA of higher eukaryotes AP sites irrespectively of their origin are thought to
be repaired by base excision repair (Almeida & Sobol, 2007; Hegde et al., 2008).
Selected Topics in DNA Repair
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2.1 Base excision repair (overview)
Base excision repair (BER) is one of the major systems of DNA repair, mostly responsible for
removing from DNA of non-bulky base lesions that appear in the genome with high
frequency (Almeida & Sobol, 2007; Hegde et al., 2008; Schärer, 2003). The base excision
repair pathway essentially removes and replaces nucleotides containing aberrant bases in
DNA. Metabolically produced reactive nitrogen and oxygen species can modify the DNA
bases due to oxidation, deamination and even alkylation at several positions in the base.
BER is usually defined as DNA repair initiated by a lesion-specific DNA glycosylase and
completed by either of two sub-pathways: short-patch BER; a mechanism whereby only 1
nucleotide is replaced or long-patch BER; a mechanism whereby 2–13 nucleotides are
replaced (Frosina et al., 1996; Klungland & Lindahl, 1997).
The majority of BER is currently thought to occur via the short-patch pathway initiated by
either a mono-functional or bi-functional DNA glycosylases. The short-patch BER pathway
mediated by a mono-functional glycosylase involves removal of aberrant base by the lesion-
specific DNA glycosylases, enzymes that hydrolyze cleavage of N-glycosylic bond between
base and deoxyribose. This results in apurinic/apyrimidinic (also termed abasic) sites (AP
sites), which repair is generally initiated through strand incision at its 5'-side by an AP
endonuclease, leaving a nick flanked by a 3'-hydroxyl of an undamaged and a deoxyriboso-
5'-phosphate (dRP), to which the damaged base was formerly linked. DNA polymerase then
inserts a normal deoxyribonucleotide; however, the ligation step to restore intact DNA is
blocked because of the dangling dRP moiety. The situation is resolved by a special
enzymatic activity, excising dRP moiety (short-patch), or by continuing DNA synthesis with
strand displacement, followed by removal of the displaced strand by flap endonuclease 1
(long-patch BER) (Frosina et al., 1996; Klungland & Lindahl, 1997).
In mammalian cells, the major dRP-removing enzyme is DNA polymerase β (Pol β), a
multifunctional enzyme consisting of the 8-kDa amino-terminal domain with deoxyribose
phosphate (dRP) lyase activity and the 31-kDa carboxy-terminal domain with nucleotidyl
transferase activity (Allinson et al., 2001, Matsumoto & Kim, 1995, Piersen et al., 1996,
Podlutsky et al., 2001). Thus, Pol β can mediate both steps in single nucleotide BER:
insertion of deoxyribonucleotide and removal of dRP moiety preparing the strand for
ligation.
Oxidized bases in DNA are manly produced by reactive oxygen species (ROS). ROS are
generated as by-products of metabolic processes, primarily oxidative metabolism in the
mitochondria (Dawson et al., 1993), and pathological conditions such as inflammation. ROS
are also generated by ionizing radiation and some chemotherapeutic drugs (Almeida &
Sobol, 2007; Hegde et al., 2008; Schärer, 2003). Oxidized bases are primarily removed by bi-
functional DNA glycosylases that have an additional AP site cleavage activity. In addition to
oxidation of DNA bases, ROS attack deoxyribose in DNA to generate strand breaks with
nonligatable ends. The 3′ blocking groups include 3′ phosphate, 3′ phosphoglycolaldehyde,
or 3′ phosphoglycolate (Breen et al., 1995). The 5′ terminus normally contains phosphate but
after ROS reaction the nonligatable ends include 5′ OH and 5′ phosphodeoxyribose
derivatives such as 2-deoxyribonolactone (Demple et al., 2002). Repair of single-strand
breaks (SSBR) with blocked termini utilizes many of the same proteins as the BER process.
The principal difference between SSBR and BER is the initiation step. Further both processes
may occur at the single nucleotide level (i.e., short-patch repair) or as a long-patch of repair.
BER is initiated via DNA glycosylase activity that results in the removal of a damaged base.
SSBR is defined specifically for the repair of single-strand breaks in DNA generated by
New Players in Recognition of Intact and Cleaved AP Sites:
Implication in DNA Repair in Mammalian Cells
307
irradiation or ROS (Fan & Wilson, 2005). AP sites appeared via spontaneous base loss and
arising from irradiation are also further processed by the BER machinery.
2.2 Processing of AP sites
AP sites can be incised by three mechanistically different ways represented in Fig.1.
Fig. 1. Structures of intact and cleaved AP sites. DNA structures containing reactive sugar
moieties capable of Schiff base formation are marked by grey rectangular boxes.
In mammalian cells, the repair of AP sites is generally initiated through strand incision by
APE1, the second enzyme of the canonical BER pathway (Demple et al., 1991). APE1 is
thought to process of over 95% of AP sites in mammalian cells (Demple & Harrison, 1994;
Wilson & Barsky, 2001). APE1 catalyzes hydrolysis of AP sites by a Mg
2+
-dependent
mechanism, resulting in cleavage of the phosphodiester bond 5’ to the AP site and
generation of a single-strand break. This reaction produces a 3’-hydroxyl group and a 5’-
dRP group flanking the break. This way of AP site processing fits short-patch pathway of
the BER process.
Another way of AP site cleavage by the BER machinery involving the action of bi-functional
DNA glycosylases is more complicated and requires the ‘end cleaning’ of termini prior to
the further repair reactions may occur. An intermediate reaction step for these enzymes is
the formation of a transient Schiff base between the amino group and the C1′ of deoxyribose
for both base excision and subsequent DNA strand cleavage. In solution, AP sites exist
predominantly as a mixture of ring-closed α- and β-hemiacetals with a minor amount of
ring-opened aldehyde and aldehyde hydrate (<1%) (de los Santos et al., 2004). AP sites can
be incised through -elimination using ε-NH2 of a lysine as the active site nucleophile
(David & Williams, 1998; McCullough et al., 1999). Strand incision by -elimination forms a
nick with phospho-α,β-unsaturated aldehyde (PUA) at the 3’ margin and phosphate at the 5’
margin (David & Williams, 1998; McCullough et al., 1999) (Fig. 1). While recently discovered
mammalian DNA glycosylases, termed NEIL (Nei-like)-1, -2, catalyze the ,δ-elimination
Selected Topics in DNA Repair
308
reaction with the N-terminal proline being used as the nucleophile (Hazra et al., 2002; Takao
et al., 2002, Das et al., 2006). The ,δ-elimination reaction results in a one nucleotide gap
flanked with phosphate groups at the 3’- and 5’ margins. The products of bi-functional DNA
glycosylase activity require the trimming of the 3’-ends to produce 3’-end hydroxyl groups
that are indispensable for DNA polymerase activity.
Most of mammalian proteins known to form the Schiff base intermediate with the AP site
appear to belong to the BER system. On the other hand, an interaction of AP DNA with
proteins not formally involved in BER (for example, human ribosomal protein S3 and
nucleoside diphosphate kinase – NM23-H2/NDP) has been well documented (Hegde et al.,
2004; Postel et al., 2000). These two proteins are able to cleave AP sites. MutY, unlike other
monofuctional DNA glycosylases, is able to interact with AP site via a Schiff base formation
(Zharkov & Grollman, 1998) without the concomitant cleavage of AP sites. In this particular
case formation of the Schiff base intermediate is considered as a mechanism for temporal
protection of AP sites (Zharkov & Grollman, 1998).
Since AP sites in DNA appear to be promiscuous in their binding to many cellular factors
and they are constantly generated in high frequency in genomic DNA, it may be important
to sequester the AP sites immediately upon formation and further process them by the way
that is most favorable for DNA integrity. Thus, interaction of cellular proteins with AP sites
might be important for their repair/temporal protection from further degradation or might
be involved in damage sensing/signaling making search of AP site reactive proteins a very
important task.
2.3 Search of new participants of recognition/processing of AP sites
The very promising approach in search for unknown players in AP site recognition is based
on a well-known propensity of deoxyribose in AP site, existing in equilibrium between
cyclic furanose and acyclic aldehyde forms, to react with amine moieties in its vicinity. The
Schiff base intermediate can be reduced by sodium borohydride (NaBH
4
) or related
compounds, forming an irreversible complex between the enzyme and DNA (David &
Williams, 1998; Piersen et al., 2000). This reaction is widely used to prove the β-elimination
reaction mechanism for the enzymes capable of AP site cleavage (David & Williams, 1998;
Piersen et al., 2000), although some proteins (e.g. MutY DNA glycosylase) can form a Schiff
base with no further β-elimination (Zharkov & Grollman, 1998). Therefore, upon searching
of new players in AP site recognition mediated by the Schiff base formation their ability to
cleave AP has to be proved. Along with intact AP site, the product of its cleavage – 3’ PUA
and 5’ dRP moieties (Fig. 1, grey rectangular boxes) are also able to form the Schiff base
intermediate with primary amino groups of proteins that allows to use these DNA in search
and identification of proteins.
2.3.1 Identification of proteins reactive to AP sites
For identification of protein cross-linked to AP DNA in cell extracts immunochemical
and/or mass-spectrometry methods can be used. Identification of proteins reactive to AP
sites by combination of cross-linking technique and mass-spectrometry is schematically
represented in Fig. 2.
In general, search and identification of proteins reactive to AP sites include the following
steps: (1) finding of cellular extract proteins forming covalent adducts with AP sites in DNA;
(2) the design of AP-DNA probe containing a functional group providing the selective
New Players in Recognition of Intact and Cleaved AP Sites:
Implication in DNA Repair in Mammalian Cells
309
Fig. 2. Workflow of identification of protein cross-linked to DNA containing intact or
cleaved AP site.
isolation of cross-linked DNA-protein products; (3) the preparative cross-linking of extract
proteins with such DNA; (4) the affinity purification of the resulted products; (5) the
identification of the protein in the covalent adduct with DNA using mass-spectrometry
methods; (6) the confirmation of the results using purified proteins and/or specific
antibodies and the known functions/interactions of the identified protein; and (7) the study
of the functional role of the revealed interactions between proteins and AP DNA. A proof-
of-principle identification of protein reactive towards AP sites in species from E. coli and S.
cerevisiae was reported in (Rieger et al., 2006).
It should be noted that used protocol includes two steps that increases the selectivity of the
method. Affinity purification of cross-linked protein was carried out on commercially
available affinity sorbents containing streptavidin, which binds the biotin residue. After
adsorption, non-specifically bound proteins were removed by a series of washings.
Additional separation of proteins was achieved by electrophoresis under denaturing
conditions by the Laemmli method (Laemmli, 1970) followed by staining proteins. The
target product was identified by the colocalization in the gel positions of the label in DNA
and the stained protein. This approach indicates that only the protein cross-linked to DNA
will be subjected to the further analysis. However, one cannot fully exclude the presence of
impurity proteins with the same electrophoretic mobility as the target product.
The identification was carried out for a number of proteins forming cross-links with AP sites
in extracts of E. coli cells at logarithmic and stationary phases of cell growth (Rieger et al.,
2006). It should be noted that authors used the previously developed non-enzymatic
approach of the creation of AP sites in DNA based on the periodate oxidation of 2,3,5,6-
tetrahydroxyhexyl phosphate precursor, which was introduced into an oligonucleotide
Selected Topics in DNA Repair
310
during the standard phosphoroamidite oligonucleotide synthesis. The periodate oxidation
allows one to obtain AP sites in an almost quantitative yield. This method, in the authors’
opinion, has several advantages over the more commonly used method that is based on the
removal of uracil residues using uracil DNA glycosylase.
The procaryotic proteins AroF, DnaK, MutM, PolA, TnaA, TufA, and UvrA from E. coli
and eucaryotic ARC1 and Ygl245wp from yeast were identified (Rieger et al., 2006).
Protein Ygl245wp with an unknown but essential for cell viability function was prepared
in the individual state after its coding sequence was cloned, and the recombinant plasmid
was used for the production of the protein in E. coli cells followed by its isolation. The
obtained protein was shown to bind to AP DNA forming Schiff bases; however, the
biological significance of this interaction has not been established. DNA polymerase I, like
other DNA polymerases of the A-family, has a weak AP lyase activity and dRP lyase
activity (Pinz & Bogenhagen, 2000). However the biological significance of these activities
in vivo has not been established. The ability of UvrA, the key component of the nucleotide
excision repair (NER) in bacteria, to interact with AP sites in the UvrABC complex
indicates the possible role of NER as a back-up pathway of AP site repair in bacteria
(Showden et al., 1990).
2.3.2 Identification of Ku80 subunit of human Ku antigen as a protein reactive to AP
sites
Using the same technique we screened mammalian cell extracts for proteins capable of
binding AP site-containing DNA. In HeLa cell extract, a 32-bp DNA duplex with an AP site
in the middle of the DNA chain was shown to cross-link predominantly to a protein forming
a product with an apparent molecular mass of 95 kDa (Fig. 3) (Ilina et al., 2008).
The subsequent analysis of whole cell extracts of human lung fibroblasts, K-562, and MCF-7
cells revealed the products with the same electrophoretic mobility. The preferential cross-
linking of this protein reflected extraordinary specificity in light of the multitude of proteins
in the cell. If the AP site in DNA is replaced by its analog (the THF residue) or the treatment
with NaBH4 is omitted, DNA–protein cross-linked products are not registered (Fig. 3).
In spite of efficient cross-linking of the above mentioned AP DNA to Ku80 polypeptide
(as a part of DNA-PK) no cleavage of AP sites was observed (Ilina et al., 2008). Instead
Ku80 formed with AP site a long-living Schiff base intermediate without the concomitant
AP site cleavage just as was observed for monofunctional DNA glycosylase MutY and
considered as a mechanism for temporal protection of AP sites (Zharkov & Grollman,
1998). AP lyase and 5’-dRP activities are distinctive features of the BER process (Almeida
& Sobol, 2007; Hegde et al., 2008). The BER 5’-dRP/AP lyases usually function beyond the
DS-breaks. But abasic sites associated with double-strand breaks can be generated by
ionizing radiation, by treatment with radiomimetic drugs or as a result of attempted BER
of complex damages (Yang et al., 2004). These specifically positioned lesions must be
removed prior to or in the course of DS break repair. Ku antigen – DS-end binding protein
of NHEJ – has been recently shown to act as a 5’-dRP/AP lyase near double-strand breaks
(Roberts et al., 2010).
At DSB ends Ku is approximately tenfold more active than Pol β (Roberts et al., 2010), the
most known mammalian 5’-dRP lyase (Matsumoto & Kim, 1995). At the same time, Ku was
inefficient as AP lyase at AP sites situated at a distance longer than one helix turn from DS
breaks just as was reported in (Ilina et al., 2008). NaBH
4
-dependent cross-linking of