Telomere overhang generation processes and the end-replication problem Telomeres are composed of TTAGGG repeated sequences located at the end of chromosomes. In human somatic cells, cell divisions are accompanied with progressive telomere length shortening due to lack of or insufficient telomerase activity (termed the end-replication problem). It is generally believed that telomere length shortening results from the removal of the RNA primer binding site at the Okazaki fragment located in lagging DNA strand synthesis. If that were correct, the rate of telomere shortening would only be around 8-10 bp per cell division (RNA primer size ÷ 2 = (16 ~ 20 nt) ÷ 2). Nevertheless, telomerase negative human cells show telomere shortening rates of ~60-70 bp per cell division. From previous studies, we found that telomere overhang generation processes may be important in determining telomere shortening rates. We found that the telomere lagging strand has an overhang of ~110 nt and the leading strand has an overhang of ~30 nt. These results can explain the telomere shortening rate in telomerase negative cells [(lagging strand overhang 110 nt + leading strand overhang 30 nt)÷2 = ~70nt]. We have recently found that human telomere overhangs in telomerase-negative cells are processed by multiple steps and leading/lagging strands are produced by distinct mechanisms. Leading strand overhangs have an early process that occurs throughout S phase, and a late process that occurs in late S/G2 phase. In contrast, lagging strand overhang processes are completed as soon as the end is replicated. At present, we are determining the molecular factors involved in overhang generation processes using biochemical approaches as well as a new system for introducing telomerase negative conditions in various human cell lines using advanced techniques of genome editing.