Visualization of uracils created by APOBEC3A using UdgX shows colocalization with RPA at stalled replication forks

Abstract

The AID/APOBEC enzymes deaminate cytosines in single-stranded DNA (ssDNA) and play key roles in innate and adaptive immunity. The resulting uracils cause mutations and strand breaks that inactivate viruses and diversify antibody repertoire. Mutational evidence suggests that two members of this family, APOBEC3A (A3A) and APOBEC3B, deaminate cytosines in the lagging-strand template during replication. To obtain direct evidence for the presence of these uracils, we engineered a protein that covalently links to DNA at uracils, UdgX, for mammalian expression and immunohistochemistry. We show that UdgX strongly prefers uracils in ssDNA over those in U•G or U:A pairs, and localizes to nuclei in a dispersed form. When A3A is expressed in these cells, UdgX tends to form foci. The treatment of cells with cisplatin, which blocks replication, causes a significant increase in UdgX foci. Furthermore, this protein- and hence the uracils created by A3A- colocalize with replication protein A (RPA), but not with A3A. Using purified proteins, we confirm that RPA inhibits A3A by binding ssDNA, but despite its overexpression following cisplatin treatment, RPA is unable to fully protect ssDNA created by cisplatin adducts. This suggests that cisplatin treatment of cells expressing APOBEC3A should cause accumulation of APOBEC signature mutations.

Base-Resolution Analysis of Deoxyuridine at the Genome Scale Based on the Artificial Incorporation Modified Nucleobase

“… 22 Moreover, several different approaches were also designed for in situ detection of uracil–DNA in the cellular context using fluorescence microscopies. 8 , 25 , 26 Nonetheless, the mapping of the distribution of uracil at genome-wide still remains a challenge besides impeding an in-depth view of global uracil events. dU-seq, 23 UPD-seq, 24 and U-DNA-Seq 8 were also developed to obtain the local information regarding the dU.…”

Detection of Genomic Uracil Patterns

“…Further developments led to two alkoxyamine reagents, AA3 and AA6, associated with increased reactivity and functional groups, appropriate to conjugate with a wide variety of biochemical labels by click chemistry [ 82 , 83 ]. These reagents were used in different applications [ 84 , 85 , 86 ].…”

“…An mCherry tagged UdgX was constructed and characterized in detail as a highly sensitive U-DNA sensor for detecting U-DNA by confocal microscopy in wild type (wt) and ung −/− dut −/− E. coli [ 96 ]. A FLAG-tagged UdgX-based sensor was also used in human cells to detect uracils in ssDNA arising upon the induction of APOBEC3A in cisplatin-treated HEK293T cells, revealing uracil colocalization with replication protein A in stalled replication forks [ 85 ]. An advantage of this sensor might be that it can be applied by transfection into living cells, and then only the immunodetection steps have to be carried out in fixed samples.…”

“…These in situ detection solutions provide potent tools within highly different biological samples for relatively quick and efficient detection of genomic uracils either upon their increasing levels (e.g., drug treatments [ 91 ]) or upon spatial clustering into genomic loci (e.g., targeted enzymatic cytosine deamination [ 85 ]). Earlier, in situ detection method for AP sites were available [ 77 ], but their application for detection of genomic uracil is not straightforward at all.…”

“…Sequencing over the crosslink might be challenging, although may not be impossible to solve. Considering that UdgX has strong preference towards uracils in ssDNA [ 85 ], and once it binds to it, other UDGs cannot initiate its conversion to AP sites anymore [ 92 ], such an approach could provide selective and safe enrichment of otherwise more vulnerable uracil-containing ssDNA fragments directly from cells. Moreover, high throughput analysis of the crosslinked peptide–DNA fragments by mass spectrometry could also provide single base resolution data wherever it is interesting.…”