Regulation of DNA replication at the end of the mitochondrial D-loop involves the helicase TWINKLE and a conserved sequence element

Abstract

The majority of mitochondrial DNA replication events are terminated prematurely. The nascent DNA remains stably associated with the template, forming a triple-stranded displacement loop (D-loop) structure. However, the function of the D-loop region of the mitochondrial genome remains poorly understood. Using a comparative genomics approach we here identify two closely related 15 nt sequence motifs of the D-loop, strongly conserved among vertebrates. One motif is at the D-loop 5′-end and is part of the conserved sequence block 1 (CSB1). The other motif, here denoted coreTAS, is at the D-loop 3′-end. Both these sequences may prevent transcription across the D-loop region, since light and heavy strand transcription is terminated at CSB1 and coreTAS, respectively. Interestingly, the replication of the nascent D-loop strand, occurring in a direction opposite to that of heavy strand transcription, is also terminated at coreTAS, suggesting that coreTAS is involved in termination of both transcription and replication. Finally, we demonstrate that the loading of the helicase TWINKLE at coreTAS is reversible, implying that this site is a crucial component of a switch between D-loop formation and full-length mitochondrial DNA replication.

Maintenance and Expression of Mammalian Mitochondrial DNA

“…It appears unlikely that the 7S RNA could actually prime DNA synthesis because the transcript is polyadenylated at its 3 end and not associated with mtDNA. Instead, we propose that the 7S RNA is the product of active termination at CSB1 (139). Ligation is a delicate process and DNA ligase III function may be disturbed by ongoing transcription.…”

“…The region between LSP and the start of the displacement loop (D-loop),is actively transcribed, producing primers for the initiation of DNA synthesis, 7S RNA transcripts, and full-length polycistronic transcripts. The function of the 7S RNA is not known, but it is mostly discussed in terms of being a primer for the initiation of DNA replication at O H (22,138,139). It appears unlikely that the 7S RNA could actually prime DNA synthesis because the transcript is polyadenylated at its 3 end and not associated with mtDNA.…”

“…Replication of mtDNA may be regulated at four different levels, at least: (a) Regulation may occur through the initiation of transcription at LSP because transcription is a prerequisite for primer formation (30); (b) Regulation may occur through termination or processing of the LSP transcript to generate a 3 end in the CSB2 region to initiate DNA replication (53,54); (c) Regulation may occur by terminating DNA synthesis after approximately 650 nucleotides to form the 7S DNA (139,143); and (d ) The regulation of the fraction of mtDNA molecules that are available for the initiation of DNA replication may occur by controlling the degree of mtDNA compaction (41,144). About two-thirds of all transcription events initiated at LSP in vitro are prematurely terminated at CSB2 by the formation of G-quadruplex structures in the nascent RNA (126,127).…”

“…The balance between abortive and genome-length mtDNA replication is, therefore, likely to be regulated at the end of the D-loop region rather than at O H (139,143). The 7S DNA resulting from abortive replication remains bound to its parental L-strand, whereas the nontemplate H-strand is displaced.…”

Biochemical Characterization of the Human Mitochondrial Replicative Twinkle Helicase

“…Collectively, the in vitro studies suggest that Twinkle may be capable of acting upon D-loop substrates by a combination of its branch migration and helicase activity, the latter not restricted to D-loops with pre-existing 5Ј singlestranded DNA tails. These findings are pertinent to the finding that Twinkle (and pol ␥) are highly enriched in the D-loop region and may be relevant to reloading of the replisome components at the end the mitochondrial D-loop (35).…”