The integrity of genomic DNA is constantly challenged by endogenous and exogenous hazards generating numerous DNA lesions in human cells daily.
These DNA lesions, if not repaired precisely and timely, can lead to genomic instability.
To deal with the risks of DNA damage, cells use a complicated molecular network to sense, signal, and repair DNA lesions, aka DNA damage response (DDR)
One of the major components of DDR involves chromatin remodeling at the DNA lesions, which is primarily achieved with histone modifications .
The most prominent histone modifications induced by DNA damage occur on H2AX, a variant of canonical histone H2A .
In response to DNA damage, especially DNA double‐strand breaks (DSBs), H2AX is rapidly phosphorylated at serine 139 (S139) by PI3‐like kinases including ATM, ATR, and DNA‐PK .
The phosphorylated H2AX is also known as γH2AX, which is recognized by the downstream mediators such as MDC1 assists in the recruitment of repair machinery for DSB repair.
In addition to phosphorylation, other posttranslational modifications (PTMs) on histones, such as ubiquitination , SUMOylation, methylation, and acetylation, also regulate chromatin status adjacent to DNA lesions and facilitate DNA damage repair.
Among all damage signals, ADP‐ribosylation is one of the earliest signals generated at DNA lesions, and histones due to their proximity with the DNA function as primary substrates of ADP‐ribosylation in response to DNA damage.
proximity[prɑːkˈsɪməti] Proximityto a place or person is nearness to that place or person.
Protein ADP‐ribosylation is catalyzed by a group of poly(ADP‐ribosyl)ation polymerases (aka PARPs) .
These enzymes utilize NAD+ as the ADP‐ribose donor and transfer ADP‐ribose moiety to protein substrates.
In human cells, 17 PARPs have been identified , and many of them are known to mediate ADP‐ribosylation in response to DNA damage .
In particular, PARP1 mediates the majority of ADP‐ribosylation in response to DNA damage .
Of note, not all the amino acid residues can accept PARP1‐mediated ADP‐ribosylation.
Mass spectrometry analyses of the substrate proteins show that glutamate (E), aspartate (D), tyrosine (Y), and serine (S) residues function as primary targets of PARP1‐mediated ADP‐ribosylation .
Recent studies have shown that other residues including lysine (K) may also be ADP‐ribosylated through alternate mechanisms.
In addition to PARP1, other PARPs, including PARP 2, 3, 7, and 10, also catalyze ADP‐ribosylation during DDR, and thus may have overlapping functions.
The general biological functions of ADP‐ribosylation in DDR have been studied extensively.
It has been shown that ADP‐ribosylation brings huge amounts of negative charge to the chromatin adjacent to DNA lesions, which may facilitate chromatin relaxation .
Moreover, several ADP‐ribosylation binding motifs have been identified, suggesting that ADP‐ribosylation, like phosphorylation, acts as a signal to mediate the recruitment of DNA damage repair machinery to DNA lesions for the repair.
However, like protein phosphorylation, different ADP‐ribosylation sites may mediate different steps of DNA damage repair .
Due to lack of analytic approaches, the biological functions of site‐specific ADP‐ribosylation in DNA damage repair are not well studied.
Fortunately, over the past few years, advanced mass spectrometry technologies have revealed more and more ADP‐ribosylation sites .
In this study, using unbiased high‐resolution mass spectrometry and other molecular biology approaches, we characterized the ADP‐ribosylation of E141 of H2AX and provided insights into its critical role in base excision repair (BER).
Oxidative stress is recognized as a potent inducer of protein ADP‐ribosylation; notably, ADP‐ribosylation would be strongly induced following higher oxidative stress.
In this study, we demonstrate that the E141 of H2AX is ADP‐ribosylated in response to oxidative damage.
Moreover, this ADP‐ribosylation site is recognized by the GRFs of Neil3, which is important for BER/SSB repair.
A unique feature of this ADP‐ribosylation site is its close to the S139 phosphorylation site on H2AX.
S139 is phosphorylated by PI3‐like kinases during DNA damage response.
Generally, γH2AX is also known as the surrogate marker of DSBs .
However, S139 of H2AX is also phosphorylated when SSBs are generated (Katsube et al, 2014).
In contrast, ADP‐ribosylation of E141 is only occurred in response to oxidative damage.
Moreover, ADP‐ribosylation of E141 and phosphorylation of S139 are mutually exclusive events.
E141的 ADP 核糖基化和 S139的磷酸化是互斥。
And suppression of ADP‐ribosylation of E141 promotes the ATM kinase‐mediated phosphorylation of S139, suggesting that ADP‐ribosylation of E141 may negatively regulate the phosphorylation of S139.
Since ADP‐ribosylation brings negatively charged phosphate moieties, it is possible that the negative charge changes the topology of the S139 motif and thereby preventing phosphorylation.
由于 ADP 核糖基化产生带负电荷的磷酸基团，可能是负电荷改变了 S139基序的拓扑结构，从而阻止了磷酸化。
Indeed, this phenomenon may facilitate SSB repair because S139 phosphorylation is directly recognized by DSB repair machinery such as MDC1.
In the event of loss in γH2AX suppression, MDC1 will accumulate at the damage sites.
Although γH2AX is known to be induced by oxidative stress, the biological function of γH2AX‐dependent pathway remains elusive.
It is possible that MDC1 might be indirectly involved in BER.
Thus, ADP‐ribosylation of E141 may indirectly facilitate SSB repair by masking the epitope of the S139 motif of H2AX and suppress any possible misloading of DSB repair factors.
In addition to its indirect role in regulating γH2AX during SSB response, E141 ADP‐ribosylation also directly participates in BER by mediating the recruitment of Neil3.
Neil3 is a unique DNA glycosylase that can excise both oxidized purines and pyrimidines, including spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), 2,6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine (FapyG), and 4,6‐diamino‐ 5‐formamidopyrimidine (FapyA) DNA lesions .
In addition to its role in BER, Neil3 is also known to participate in DNA interstrand crosslinks (ICLs) repair (Liu et al, 2013; Zhou et al, 2017; Wu et al, 2019).
However, we found that H2AX ADP‐ribosylation is not induced in by ICLs, indicating that H2AX ADP‐ribosylation‐mediated recruitment of Neil3 may not be involved in ICLs repair.
Also our results show that H2AX is oligo(ADP‐ribosyl)ated in response to oxidative damage.
In addition to binding to the ADP‐ribose units, Neil3 is likely to recognize other motifs on H2AX, which determines the specificity of this particular ADP‐ribosylation site on chromatin and exclusive function of this ADP‐ribosylation event in BER.
Future structural analysis will reveal the detailed binding between Neil3 and ADP‐ribosylated H2AX.
Although γH2AX formation happens during SSB repair, it might be a slightly late stage event (Driessens et al, 2009; Katsube et al, 2014).
It has been shown that γH2AX may participate in oxidative stress‐induced SSB repair at stalled replication forks (Katsube et al, 2014). However, ADP‐ribosylation of H2AX may be a much earlier event. Consistently, we have shown that phosphorylation and ADP‐ribosylation are mutually exclusive, although these two modifications locate close to each other.
Similar to the S‐Q‐E motif in H2AX, there are many other SQE/SQD motifs that can be phosphorylated by PI3‐like kinases.
Interestingly, the +2 position E or D may also the acceptors for ADP‐ribosylation.
Thus, the functional interaction between phosphorylation and ADP‐ribosylation may occur in other DNA repair mediators/effectors.
Here, our results provide the first evidence of such functional interactions between these two important posttranslational modifications during SSB repair process.
In addition, the crosstalk between ADP‐ribosylation and phosphorylation can be different in other types of damage response, such as DSBs response.
In response to DSBs, phosphorylation of H2AX is clearly observed but not the ADP‐ribosylation.
And others have noticed that the E141A mutation may slightly change the epitope of S139 motif and thus affect the robust phosphorylation of H2AX by the PI3‐like kinases during DSB repair (Rogakou et al, 1998; Burma et al, 2001; Celeste et al, 2003).
Because of the different damage response, it would be difficult to directly compare the crosstalk between ADP‐ribosylation and phosphorylation of H2AX.
Collectively, our study reveals that E141 of H2AX is ADP‐ribosylated in response to DNA base damage.
Moreover, we demonstrate molecular mechanism by which this posttranslational modification event regulates BER/SSB.
https://www.embopress.org/doi/full/10.15252/embj.2020104542#:~:text=Here%2C%20ADP%E2%80%90ribosylation%20of%20histone%20variant%20H2AX%20on%20a,histone%20H2AX%20is%20ADP%E2%80%90ribosylated%20following%20oxidative%20DNA%20damage.
