Subsequently, we considered twin priming, a mechanism that did not at first appear to be consistent with the patterns we observed in our loci. This mechanism results in L1 inversions accompanied by internal deletions to the L1 sequence [, , ]. In this mechanism, the L1 mRNA anneals using its poly(A) tail to the bottom strand EN nick site and an RT primes at this location and begins to synthesize the L1 cDNA exactly as in classical TPRT (Figure ). However, once the top strand is nicked, generating a 3' overhang, this model proposes that a position internal to the mRNA may anneal to the overhang, allowing a second RT molecule to prime and begin synthesizing cDNA in the antisense orientation on the top strand. The resulting twin priming insertion is characterized by TSDs bounding two inverted fragments of the same L1 and containing an internal deletion of the L1 sequence (Figure ). An assumption of the twin priming mechanism is that the second strand nick must occur before first strand reverse transcription is completed [, ].
Investigation of candidate loci and variations within the homopolymeric stretches. (a) A triple alignment of pT684 to two outgroup species, the rhesus macaque and the common marmoset. The TSDs are highlighted in grey, the poly(T) stretch in green, and the L1 is highlighted in blue. (b) A gel chromatograph of polymerase chain reaction products depicting an insertion present in humans, chimpanzees, gorillas and orangutans, but absent in rhesus macaque and owl monkey. (c) Internal primers were designed around the poly(T) stretches for all human-specific loci; two loci are shown here. For each locus, HeLa DNA and a mixture of the DNA of 80 human individuals was run out on a 4% agarose gel with 100 bp and 20 bp ladders. No within-species variation in poly(T) length was observed.
DNA polymerase I is the predominant polymerizing enzyme found in E. coli. It contains a single disulfide bond and one sulfhydryl group (Jovin et al. 1969b). Five distinct DNA polymerases have been isolated from E. coli and have been designated I, II, III, IV, and V. DNA polymerase I functions to fill DNA gaps that arise during DNA replication, repair, and recombination. DNA polymerase II also functions in editing and proofreading mainly in the lagging strand (Kim et al. 1997, Wagner and Nohmi 2000). DNA polymerase III is the main replicative enzyme. DNA polymerase IV and V have large active sites that allow for more base misincorporation, and are therefore more error-prone. They also lack proofreading-exonuclease subunits to correct misincorporations (Nohmi 2006, and Hastings et al. 2010). DNA polymerase V is present at significant levels only in SOS-induced cells and over-expression restricts DNA synthesis (Marsh and Walker 1985).
The RNA Polymerase moves stepwise along the DNA, unwinding the DNA helix just ahead of the active site for polymerization to expose a new region of the template strand for complementary base-pairing.
DNA synthesis requires a primer strand with a free 3’-hydroxyl terminus annealed to a DNA template strand and the deoxynucleotide triphosphates form base pairs with the template. Addition is in the 5’ to 3’ direction with release of pyrophosphate. The enzyme is active with DNAs containing single stranded gaps and also with DNAs with single-strand breaks or nicks. Under some conditions, RNA-DNA hybrids and an RNA duplex may serve as template-primer (Setlow 1972).
The target site analyses and microhomology results we obtained implicate a variant of TPRT as the mechanism generating these loci. We found significant microhomology at the 5' end of the poly(T) stretch and the 3' end of the L1 insertion. Interestingly, it is not the 3' target site that closely resembles the canonical L1 EN cleavage site, but the complementary sequence of the 5' target site nearest the stretch of poly(T)s. As described above, our analysis of the reverse-complemented sequence adjacent to the poly(T) stretch recovered no evidence of inverted L1 sequence at this junction. While previous twin priming studies found some microhomology at the internal junction, this was usually less than that found at the target site, and in some cases, no microhomology was found [, ]. One explanation that may account for this appearance involves the poly(A) tail of the element being reverse transcribed, but assumes that this first RT disengages prior to exiting the tail and entering the L1 sequence proper. The other priming event, occurring internally on the mRNA, then synthesizes a portion of the L1 cDNA. When viewed with the candidate L1 in the sense orientation, the poly(A) tail is reverse complimented, forming a stretch of poly(T)s located 5' to the L1 (Figure ). To determine if a short portion of non-inverted L1 sequence was found after the poly(T) stretches, a simple check involving an alignment of the reverse complement of the poly(T) stretch and following 50 bp of our insertions to an L1 consensus could find no match to the 3' end of the consensus.
In 1969, Jovin et al. elucidated the amino acid composition (Jovin et al. 1969a, b). That same year, DeLucia and Cairns isolated an E. coli strain with a mutation that affected the DNA polymerase and surprisingly found that the mutant synthesized DNA normally. This discovery cast doubts on the role of DNA polymerase in replication and led groups to search for other replication enzymes. At the same time, Klenow and colleagues showed that the treatment of DNA polymerase with the proteolytic enzyme subtilisin (type Carlsberg) resulted in an increase of polymerase activity and decrease of exonuclease activity. The resulting DNA polymerase was isolated and was named the “Klenow fragment” (Klenow and Henningsen 1970a, and Klenow and Overgaard-Hansen 1970).
Classical target primed reverse transcription (TPRT), twin priming, variants of twin priming and dual priming mechanisms. (a) A schematic of classical TPRT. The poly(A) tail of an L1 mRNA anneals to the target site created by L1 endonuclease. L1 reverse transcription (RT) primes at the target site and synthesizes the bottom-strand cDNA. A subsequent second-strand nick and synthesis results in an L1 insertion with a 3' poly(A) flanked by TSDs. (b) Twin priming. In this variant of TPRT, after the second-strand nick, a site internal to the mRNA anneals to the top strand overhang. A second RT molecule primes at this site, generating an inverted L1 cDNA. (c) This twin priming variant involves the disengagement of the first RT before reaching the end of the poly(A) tail, resulting in an insertion with a 5' poly(T) stretch, but lacking a 3' poly(A) tail. Like classical twin priming, this mechanism results in an inverted L1 structure. (d) A second twin priming variant creates an insertion with both a 3' poly(A) tail and a 5' poly(T) stretch. The first RT falls off before reaching the end of the poly(A) tail. (e) Dual priming. Classical TPRT involving the first mRNA begins on the first strand. After the second strand nick, a second mRNA anneals to the second strand and undergoes classical TPRT. Note that this panel is rotated 180° relative to the orientation of all other panels. This is done to show that the resulting insertion will appear the same to computational filters as the above twin priming variant.