Coding constraints modulate chemically spontaneous mutational replication gradients in mitochondrial genomes.

Distances from heavy and light strand replication origins determine duration mitochondrial DNA remains singlestranded during replication. Hydrolytic deaminations from A->G and C->T occur more on single- than doublestranded DNA. Corresponding replicational nucleotide gradients exist across mitochondrial genomes, most at 3rd, least 2(nd) codon positions. DNA singlestrandedness during RNA transcription causes gradients mainly in long-lived species with relatively slow metabolism (high transcription/replication ratios). Third codon nucleotide contents, evolutionary results of mutation cumulation, follow replicational, not transcriptional gradients in Homo; observed human mutations follow transcriptional gradients. Synonymous third codon position transitions potentially alter adaptive off frame information. No mutational gradients occur at synonymous positions forming off frame stops (these adaptively stop early accidental frameshifted protein synthesis), nor in regions coding for putative overlapping genes according to an overlapping genetic code reassigning stop codons to amino acids. Deviation of 3rd codon nucleotide contents from deamination gradients increases with coding importance of main frame 3rd codon positions in overlapping genes (greatest if these are 2(nd) position in overlapping genes). Third codon position deamination gradients calculated separately for each codon family are strongest where synonymous transitions are rarely pathogenic; weakest where transitions are frequently pathogenic. Synonymous mutations affect translational accuracy, such as error compensation of misloaded tRNAs by codon-anticodon mismatches (prevents amino acid misinsertion despite tRNA misacylation), a potential cause of pathogenic mutations at synonymous codon positions. Indeed, codon-family-specific gradients are inversely proportional to error compensation associated with gradient-promoted transitions. Deamination gradients reflect spontaneous chemical reactions in singlestranded DNA, but functional coding constraints modulate gradients.