Multiple Mechanisms Contribute To Telomere Maintenance

Abstract

The unlimited growth potential of tumors depends on telomere maintenance and typically depends on telomerase, an RNA-dependent DNA polymerase, which reverse transcribes the telomerase RNA template, synthesizing telomere repeats at the ends of chromosomes. Studies in various model organisms genetically deleted for telomerase indicate that several recombination-based mechanisms also contribute to telomere maintenance. Understanding the molecular basis of these mechanisms is critical since some human tumors form without telomerase, yet the sequence is maintained at the telomeres. Recombination-based mechanisms also likely contribute at some frequency to telomere maintenance in tumors expressing telomerase. Preventing telomere maintenance is predicted to impact tumor growth, yet inhibiting telomerase may select for the recombination-based mechanisms. Telomere recombination mechanisms likely involve altered or unregulated pathways of DNA repair. The use of some DNA damaging agents may encourage the use of these unregulated pathways of DNA repair to be utilized and may allow some tumors to generate resistance to these agents depending on which repair pathways are altered in the tumors. This review will discuss the various telomere recombination mechanisms and will provide rationale regarding the possibility that L1 retrotransposition may contribute to telomere maintenance in tumors lacking telomerase.

Keywords: DNA repair; Retrotransposons; Telomere; Tumors.

Figure 1. Break-induced replication

Shown are models of break-induced replication based on studies in yeast (54). A. For Type II survivors, the 3’-OH of a short telomere (green) strand invades into another telomere repeat. This sequence can be on the other homolog or on a different chromosome. Strand invasion generates a replication fork and a Holliday junction. Conventional DNA polymerases are used to synthesize both strands, followed by resolution of the Holliday junction. BIR that initiates in the telomere repeat will result in a net gain of telomere sequence. B. For Type I survivors, BIR in subtelomere repeats occurs when telomere shortening proceeds into a Y’ (blue) repeat. BIR that is initiated within the subtelomeres results in a net gain in Y’ sequence.

Figure 2. Assays to directly examine telomere recombination

A. CO-FISH assay: Replicating cells are grown in the presence of the nucleotide analogs BrdU/C, for one complete round of replication. Due to semi-conservative DNA replication, BrdU/C will be incorporated into the newly replicated strand. Metaphase spreads are treated with Hoechst and UV, which nicks the BrdU/C incorporated daughter strand, which is then degraded by exonulcease III (ExoIII). Telomere probes (5’CCCTAA) are then hybridized to the ends. If a sister chromatid exchange occurred then the strand will be protected from degradation and two signals will be detected. B. pq-ratio assay: This assay measures the variation in telomere lengths at the p- and q-arms of a chromosome. The telomeres at both ends of a given chromosome shorten at a similar rate, therefore the telomere ratio for most chromosomes is expected to be q/p~1. If instead recombination is used to maintain the ends, then the amount of lengthening at one end could be different compared to the other end. This results in variable values for the pq-ratio.

Figure 3. TPRT & ENi-retrotransposition

A. A full-length retrotransposition competent L1 contains an L1-specific promoter, two intact open reading frames (ORF1 and ORF2), a polyadenylation signal sequence, and is followed by a poly(A)tail. ORF1 encodes for a nucleic acid binding protein and ORF2 encodes for both an endonuclease and a reverse transcriptase, and a C-domain of unknown function. B. Target-primed reverse transcription is a mechanism whereby the L1-encoded endonuclease cleaves the genomic DNA at a consensus cleavage site. The 3’-OH on the nicked DNA is used to prime reverse transcription of the L1 RNA. ENi-retrotransposition can also occur, whereby L1 can insert at DNA lesions or deprotected telomeres. For both conventional TPRT and ENi-retrotransposition, reverse transcription generates an intermediate that could be used during BIR.

Figure 4. BIR coupled with target-primed reverse transcription (TPRT)

ENi-retrotransposition or TPRT at a dysfunctional telomere will generate by reverse transcription a free-end that could be used to strand invade by BIR. The newly generated cDNA, would strand invade into another chromosome where a non-LTR retrotransposon resides. Conventional polymerases would copy sequences onto the chromosome using the other chromosome as a template. Thus ENi-retrotransposition or conventional TPRT could be coupled with BIR.