Powerful mutators lurking in the genome - PubMed (original) (raw)

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Powerful mutators lurking in the genome

Vincent Petit et al. Philos Trans R Soc Lond B Biol Sci. 2009.

Abstract

The human genome encodes numerous enzymes capable of deaminating polynucleotides. While they are capable of exquisite specificity, occasionally they result in hypermutation where up to 90 per cent of cytidine or adenosine residues may be edited. As such, they constitute a formidable anti-viral barrier, for no virus can survive such a high mutation rate. As the APOBEC3 group of cytidine deaminases edit single-stranded viral DNA, the crucial question is can they hyperedit chromosomal DNA? Everything points to a positive answer. Nonetheless, hypermutants per se have not yet been described, probably being countered by highly efficient mismatch repair. For the APOBEC3 genes, not only is their physiological function unknown, but also their role in the induction of cancer remains to be determined. Yet given the pace of research, all this is certain to change in the next few years.

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Figures

Figure 1

Figure 1

Selective amplification of AT- and GC-rich DNA. (a) PCR amplification of molecular clones of HIV-1 differing by between 0 and 18 G→A transitions at the non-restrictive and restrictive temperatures of 95 and 83°C, respectively. At 83°C, only clones containing G→A transitions could be amplified. A sample of a HIVΔ_vif_ virus from a molecular clone transfected on to 293T cells and subsequently used to infect PBMCs (peripheral blood mononuclear cells) was also amplified at 83°C. Cloning and sequencing showed the genomes to be G→A hypermutants resulting from APOBEC3G editing of viral cDNA. (b) 3DIPCR amplification of measles virus RNA produced in an interferon type I (i) insensitive (Vero) and (ii) sensitive (MRC5) cell line. (iv) For the MRC5 culture, DNA products could be recovered using a Td as low as 65°C, compared with (iii) the Vero control (67.4°C). Cloning and sequencing showed that the lower temperature products represent measles virus genomes edited by ADAR-1L.

Figure 2

Figure 2

Chemistry of nucleotide deamination. (a) The products of APOBEC and ADAR deamination are uridine and inosine: (i) cytidine deamination C→U and (ii) adenine deamination A→I. (b) To enable PCR-based amplification of GC-rich DNA, two non-natural dNTPs are used. (i) Diaminopurine is an adenosine analogue and base-pairs with thymidine via three hydrogen bonds. (ii) Inosine pairs mainly with cytidine via two hydrogen bonds. Exploration of the PCR denaturation temperature allows selective amplification of GC-rich DNA, which in the form of TCID DNA, as opposed to TCGA DNA, melts at lower temperatures.

Figure 3

Figure 3

AID ‘only’ edited transcribed human Vk1 sequences. (a) A single representative example is given. (b) Statistics of seven sequences bearing GC→AT transitions only. (c) The nucleotide context of the 38 GC→AT transitions resembles closely that of AID, which is WRCW, where W=A,T.

Figure 4

Figure 4

Phylogenic relationships among human APOBEC3 cytidine deamination domains (CDDs). The SplitsTree was made using protein sequences with APOBEC2 (hA2) and AID given as outliers. The CDD domains of the double-domain hA3s have been split and analysed as hA3Bn, hA3Bc, etc. where n and c indicate the N- and C-terminal domains, respectively. Those that edit HPV DNA have been highlighted.

Figure 5

Figure 5

APOBEC3 editing of HPV DNA in vivo. (a) Sample HPV1a.1. A small selection of HPV1a-edited DNA from a planter wart. Only sequence differences are given with respect to the unedited reference sequence. As only a fraction of the sequences are shown, the numbers to the right indicate the total number of mutations per sequence. (b) Dinucleotide context of HPV1a-edited sites where the dot indicates the edited site. The data for A3A, A3C and A3H were derived from transfection experiments. (c) Sample HPV16.29. A small selection of HPV16-edited DNA from a pre-cancerous cervical biopsy. Legend same as figure 5_a_. The TATA box within the promoter region is highlighted. (d) Dinucleotide context of HPV16-edited sites where the dot indicates the edited site. Black bar, experimental; grey bar, in vivo.

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