Mobility Mechanisms

All characterized mobile group II introns use a mobility mechanism in which the intron RNA in the homing endonuclease uses its ribozyme activity to reverse splice into a DNA target site and is then reverse transcribed by the IEP (reviewed in Lambowitz and Zimmerly 2004). Different variations of this mechanism are used both for "retrohoming" to a specific target site, and for "retrotransposition" to ectopic sites. For retrohoming, group II introns that encode proteins with an En domain use this domain after reverse splicing to cleave the opposite DNA strand a short distance downstream from the in-tron-insertion site (Fig. 3a). For the LLLtrB and yeast mtDNA introns, this cleavage occurs at either 3'-exon position +9 (LLLtrB; Matsuura et al. 1997) or +10 (yeast introns; Zimmerly et al. 1995a; Yang et al. 1996). The 3' end of the cleaved strand is then used as a primer for reverse transcription of the inserted intron RNA. In the case of the lactococcal intron, the resulting intron cDNA is integrated by a DNA repair mechanism independent of RecA function (Mills et al. 1997; Cousineau et al. 1998), while, for the yeast mtDNA introns, DNA recombination mechanisms play a greater role in cDNA integration (Eskes et al. 1997, 2000). Notably, yeast mtDNA introns with mutations that inhibit RT activity can still home by using the En domain to cleave the a En : Cleaved bottom strand primer

DNA Target

Intron RNA

DNA Target

Intron RNA

b tn": Nascent lagging strand primer

Intron RNA

Intron RNA

En : Nascent leading strand primer

Intron RNA

Bliii

Fig. 3. Group II intron mobility mechanisms. a Retrohoming by group II introns that encode proteins containing a C-terminal En domain. The excised intron RNA in the homing endo-nuclease reverse splices into one DNA strand, while the IEP cleaves the opposite strand and uses the cleaved 3' end as a primer for reverse transcription of the inserted intron RNA. b, c Retrohoming by group II introns that encode proteins lacking En activity. In b, the intron RNA reverse splices into transiently single-stranded DNA at a replication fork and uses a nascent lagging strand as the primer for reverse transcription. In c, the intron RNA reverse splices into double-stranded DNA prior to passage of a replication fork and uses a nascent leading strand as the primer. Thick black and gray lines indicate the 5'-exon (5'E) and 3'-exon (3'E) DNA, respectively. Thin black lines indicate intron RNA, and gray dashes indicate future cDNA. Black arrows indicate the direction of DNA replication, and gray arrows indicate newly synthesized DNA

DNA target site, thereby initiating double-strand break repair (DSBR) recombination, analogous to the mechanism of group I intron mobility (Eskes et al. 1997,2000).

Mobile group II introns that encode proteins lacking the En domain also initiate mobility by reverse splicing into DNA sites, but are unable to carry out second-strand cleavage (Muñoz-Adelantado et al. 2003). Recent studies with the Sinorhizobium meliloti Rmlntl intron indicate that a major mechanism involves the use of a nascent lagging strand at a DNA replication fork as the primer for reverse transcription (Fig. 3b; Martínez-Abarca et al. 2004). A similar mechanism has been proposed for retrotransposition of the LLLtrB intron in L. lactis (Ichiyanagi et al. 2002,2003). In both cases, the use of nascent lagging strand primers may be facilitated by reverse splicing into transiently single-stranded DNA at a replication fork. En~ mutants of the Ll.LtrB intron retrohome at decreased but still appreciable frequencies by a similar mechanism, involving reverse splicing into double-strand DNA with preferred use of nascent leading strands as primers (Fig. 3c; Zhong and Lambowitz 2003). This orientation bias is thought to reflect that, after reverse splicing into double-stranded DNA, the intron RNA is positioned to directly use a leading-strand primer, without requiring passage of the replication fork through the region containing the inserted RNP. Additionally, it has been suggested, but not demonstrated, that En~ introns might also retrohome by using non-specific opposite strand nicks to prime reverse transcription (e.g., see Schäfer et al. 2003). Group II intron mobility pathways are described in greater detail in Lambowitz and Zimmerly (2004).

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