Ci Ji Lim

Human telomere replication and maintenance

Telomeres safeguard the integrity of eukaryotic genomes. They provide a sacrificial buffer to solve the DNA end-replication problem of linear chromosomes. Telomeres also protect chromosome ends from being misrecognized as broken DNA ends. Without telomeres, mammalian cells suffer genome instability, resulting in diseases such as cancer or premature aging. Mammalian telomeric DNA is composed of thousands of tandem repeats of the conserved hexameric sequence, TTAGGG (Figure 1). The number of telomere repeats defines the telomere length and profoundly impacts cell biology.

The long-term goal of the Lim research group is to understand how mammalian telomeres safeguard the genome. To achieve this, we aim to achieve a multidimensional understanding of the mechanisms governing telomere DNA replication and maintenance in humans. This encompasses both a mesoscale comprehension of telomere chromatin’s structure and function, as well as an in-depth understanding of the molecular intricacies of telomere macromolecular assemblies and functions. Specifically, we work on three major directions as described below:

1. Structure and Function Studies of the Human Telomere DNA Replication Machinery
Central to telomere maintenance is the synthesis of telomere DNA repeats, a process delineated by two distinct pathways: telomere G-strand elongation followed by C-strand fill-in (Figure 2). Our focus is on elucidating the structural basis of shelterin regulation concerning telomerase recruitment and the telomere C-strand fill-in pathways. We employ a combination of biochemistry, genetics, and structural biology approaches to conduct structure-function studies of human shelterin-telomere and CST-polα-primase co-complexes. Insights into molecular interactions derived from these high-resolution molecular structures augment our understanding of telomere maintenance at the chromatin scale.

Compared to its G-strand counterpart synthesized by telomerase, the structural biology of mammalian telomere C-strand synthesis has not been explored as extensively. Recently, we successfully delineated one of the first cryo-EM structures of the human telomere C-strand fill-in machinery — a preinitiation state (PIC) involving CST-polα-primase bound to a telomere template (He et. al., Nature, 2022). This structure demonstrates how CST binds to the telomere DNA template, collectively acting as a scaffold to assist polα-primase in reorganizing its RNA and DNA catalytic domains, thereby facilitating the correct synthesis order of the chimeric RNA-DNA primer. In alignment with our objectives, we have also resolved the cryo-EM structures of the polα-primase elongation complexes, capturing the enzyme in the act of extending the RNA primer with DNA (He et. al., Nature Structural Molecular Biology, 2023).

2. Deciphering the Telomere Chromatin Landscape in Human Cells and its Connections with Telomere Replication and Chromosome End-Protection
Telomeres are unique due to their repetitive DNA sequences and are regulated by specific telomere-associated proteins. The binding of these proteins to telomeres is crucial for maintaining their functional state, including their length. Variations in telomere length are implicated in numerous medical conditions. Despite their importance, our understanding of how telomere-associated proteins regulate telomere length and function is limited. This knowledge gap exists largely because current techniques for studying protein-DNA interactions are not well-suited for the repetitive sequences in telomeres. As a result, we have no insight into the distribution of proteins along telomeres, which may be crucial for regulating telomere structure and length.

We aim to bridge this knowledge gap, which can potentially open a new research direction in telomere biology. The first aim is to develop innovative methodologies tailored for high-resolution mapping of the chromatin landscape of telomeres (Figure 3). The second aim is to utilize these new tools to investigate the role of protein distribution in telomere length and end-protection. We hypothesize that the unique repetitive DNA sequences in telomeres enable dynamic protein interactions that modulate telomere function and length. The innovativeness of this research direction is acknowledged by the NIH Director’s Office New Innovator Award and Pew Biomedical Scholarship.

3. Single-molecule Studies of the Mechanisms of Telomere Chromatin Assembly and Organization for Chromosome End-Protection and Telomerase Recruitment
The shelterin complex, comprising six proteins, is pivotal in regulating telomere functions including chromosome end-protection and telomerase regulation. It executes these functions through DNA-binding subunits: TRF1 and TRF2, which bind to double-stranded telomere DNA, and POT1, which interacts with the single-stranded telomere overhang. The repetitive structure of telomeric DNA, presenting thousands of similar binding sites, poses a significant challenge in understanding the regulatory role of shelterin in telomerase recruitment and the assimilation of the telomere overhang into the double-stranded DNA segment. Conventional biochemical approaches fall short in exploring these complexities.

To address this, our research group employs single-molecule biophysical techniques to probe the shelterin assemblies on elongated, repetitive telomeric DNA (Figure 4). This approach aims to delineate how these assemblies and their dynamics correlate with the regulatory mechanisms governing telomerase recruitment and T-loop formation. We utilize Atomic Force Microscopy (AFM) for single-molecule imaging to scrutinize the binding patterns of recombinant human shelterin proteins and complexes on extensive telomere DNA constructs (>10 kbp), facilitating high spatial resolution analysis of shelterin interactions with telomeric DNA. Furthermore, to unpack the dynamics of telomeric protein assembly and organization, we employ C-Trap, a tool that integrates optical tweezers and confocal fluorescence imaging, allowing us to stretch a single telomeric DNA and observe the binding and movement of fluorescently labeled telomeric DNA-binding proteins along the DNA.