Accurate DNA replication during S‐phase is fundamental to maintain genome integrity. During this critical process, replication forks frequently encounter obstacles that impede their progression. While the regulatory pathways which act in response to exogenous replication stress are beginning to emerge, the mechanisms by which fork integrity is maintained at naturally occurring endogenous replication‐impeding sequences remains obscure. Notably, little is known about how cells replicate through special chromosomal regions containing structured non‐B DNA, for example, G4 quartets, known to hamper fork progression or trigger chromosomal rearrangements. Here, we have investigated the role in this process of the human translesion synthesis (TLS) DNA polymerases of the Y‐family (pol η, pol ι, and pol κ), specialized enzymes known to synthesize DNA through DNA damage. We show that depletion by RNA interference of expression of the genes for Pol η or Pol κ, but not Pol ι, sensitizes U2OS cells treated with the G4‐tetraplex interactive compound telomestatin and triggers double‐strand breaks in HeLa cells harboring multiple copies of a G‐rich sequence from the promoter region of the human c‐MYC gene, chromosomally integrated as a transgene. Moreover, we found that downregulation of Pol κ only raises the level of DSB in HeLa cells containing either one of two breakage hotspot structured DNA sequences in the chromosome, the major break region (Mbr) of BCL‐2 gene and the GA rich region from the far right‐hand end of the genome of the Kaposi Sarcoma associated Herpesvirus. These data suggest that naturally occurring DNA structures are physiological substrates of both pol η and pol κ. We discuss these data in the light of their downregulation in human cancers. © 2008 Wiley‐Liss, Inc.